BBA - General Subjects (v.1850, #4)

Characterisation of chlorate reduction in the haloarchaeon Haloferax mediterranei by Rosa María Martínez-Espinosa; David J. Richardson; María José Bonete (587-594).
Haloferax mediterranei is a denitrifying haloarchaeon using nitrate as a respiratory electron acceptor under anaerobic conditions in a reaction catalysed by pNarGH. Other ions such as bromate, perchlorate and chlorate can also be reduced. Hfx. mediterranei cells were grown anaerobically with nitrate as electron acceptor and chlorate reductase activity measured in whole cells and purified nitrate reductase.No genes encoding (per)chlorate reductases have been detected either in the Hfx. mediterranei genome or in other haloarchaea. However, a gene encoding a chlorite dismutase that is predicted to be exported across the cytoplasmic membrane has been identified in Hfx. mediterranei genome. Cells did not grow anaerobically in presence of chlorate as the unique electron acceptor. However, cells anaerobically grown with nitrate and then transferred to chlorate-containing growth medium can grow a few generations. Chlorate reduction by the whole cells, as well as by pure pNarGH, has been characterised. No clear chlorite dismutase activity could be detected. Hfx. mediterranei pNarGH has its active site on the outer-face of the cytoplasmic membrane and reacts with chlorate and perchlorate. Biochemical characterisation of this enzymatic activity suggests that Hfx. mediterranei or its pure pNarGH could be of great interest for waste water treatments or to better understand biological chlorate reduction in early Earth or Martian environments.Some archaea species reduce (per)chlorate. However, results here presented as well as those recently reported by Liebensteiner and co-workers [1] suggest that complete perchlorate reduction in archaea follows different rules in terms of biological reactions.
Keywords: Halophile; Archaeon; Respiratory nitrate reductase; Chlorate reduction; Anaerobic respiration; Chlorite dismutase;

Electroporation is a method of choice to transform living cells. The ability of electroporation to transfer small or large chemicals across the lipid bilayer membrane of eukaryotic cells or Gram-negative bacteria relies on the formation of transient pores across the membrane. To exist, these pores rely on an insulator (the bilayer membrane) and the presence of a potential difference on either side of the membrane mediated by an external electric field. In Gram-positive bacteria, however, the wall is not an insulator but pores can still form when an electric field is applied. Past works have shown that the electrostatic charge of teichoic acids, a major wall component; sensitizes the wall to pore formation when an external electric field is applied. These results suggest that teichoic acids mediate the formation of defects in the wall of Gram-positive bacteria.We model the electrostatic repulsion between teichoic acids embedded in the bacterial wall composed of peptidoglycan when an electric field is applied. The repulsion between teichoic acids gives rise to a stress pressure that is able to rupture the wall when a threshold value has been reached. The size of such small defects can diverge leading to the formation of pores.It is demonstrated herein that for a bonding energy of about ~ 1 − 10  k B T between peptidoglycan monomers an intra-wall pressure of about ~ 5 − 120  k B T/nm3 generates spherical defects of radius ~ 0.1 − 1 nm diverging in size to create pores.The electrostatic cavitation of the bacterial wall theory has the potential to highlight the role of teichoic acids in the formation pores, providing a new step in the understanding of electroporation in Gram-positive bacteria without requiring the use of an insulator.
Keywords: Gram-positive bacteria; Biophysics; Electroporation;

The defense substance allicin from garlic permeabilizes membranes of Beta vulgaris, Rhoeo discolor, Chara corallina and artificial lipid bilayers by Martin C.H. Gruhlke; Birgit Hemmis; Ulrike Noll; Richard Wagner; Hinrich Lühring; Alan J. Slusarenko (602-611).
Allicin (diallylthiosulfinate) is the major volatile- and antimicrobial substance produced by garlic cells upon wounding. We tested the hypothesis that allicin affects membrane function and investigated 1) betanine pigment leakage from beetroot (Beta vulgaris) tissue, 2) the semipermeability of the vacuolar membrane of Rhoeo discolor cells, 3) the electrophysiology of plasmalemma and tonoplast of Chara corallina and 4) electrical conductivity of artificial lipid bilayers.Garlic juice and chemically synthesized allicin were used and betanine loss into the medium was monitored spectrophotometrically. Rhoeo cells were studied microscopically and Chara- and artificial membranes were patch clamped.Beet cell membranes were approximately 200-fold more sensitive to allicin on a mol-for-mol basis than to dimethyl sulfoxide (DMSO) and approximately 400-fold more sensitive to allicin than to ethanol. Allicin-treated Rhoeo discolor cells lost the ability to plasmolyse in an osmoticum, confirming that their membranes had lost semipermeability after allicin treatment. Furthermore, allicin and garlic juice diluted in artificial pond water caused an immediate strong depolarization, and a decrease in membrane resistance at the plasmalemma of Chara, and caused pore formation in the tonoplast and artificial lipid bilayers.Allicin increases the permeability of membranes.Since garlic is a common foodstuff the physiological effects of its constituents are important. Allicin's ability to permeabilize cell membranes may contribute to its antimicrobial activity independently of its activity as a thiol reagent.Display Omitted
Keywords: Allicin; Antimicrobial; Membranes; Lipid bilayers; Electrophysiology;

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a pathway by which misfolded or improperly assembled proteins in the ER are directed to degradation. The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme that cleaves N-glycans from misfolded glycoproteins during the ERAD process. The mutant form of yeast carboxypeptidase Y (CPY*) is an ERAD model substrate that has been extensively studied in yeast. While a delay in the degradation of CPY* in yeast cells lacking the cytoplasmic PNGase (Png1 in yeast) was evident, the in vivo action of PNGase on CPY* has not been detected.We constructed new ERAD substrates derived from CPY*, bearing epitope tags at both N- and C-termini and examined the degradation intermediates observed in yeast cells with compromised proteasome activity.The occurrence of the PNGase-mediated deglycosylation of intact CPY* and its degradation intermediates was evident. A major endoproteolytic reaction on CPY* appears to occur between amino acid 400 and 404.The findings reported herein clearly indicate that PNGase indeed releases N-glycans from CPY* during the ERAD process in vivo.This report implies that the PNGase-mediated deglycosylation during the ERAD process may occur more abundantly than currently envisaged.
Keywords: peptide:N-glycanase; ER-associated degradation; carboxypeptidase Y; Saccharomyces cerevisiae; Endoproteolysis;

Post-translational modifications on yeast carbon metabolism: Regulatory mechanisms beyond transcriptional control by Farida Tripodi; Raffaele Nicastro; Veronica Reghellin; Paola Coccetti (620-627).
Yeast cells have developed a variety of mechanisms to regulate the activity of metabolic enzymes in order to adjust their metabolism in response to genetic and environmental perturbations. This can be achieved by a massive reprogramming of gene expression. However, the transcriptional response cannot explain the complexity of metabolic regulation, and mRNA stability regulation, non-covalent binding of allosteric effectors and post-translational modifications of enzymes (such as phosphorylation, acetylation and ubiquitination) are also involved, especially as short term responses, all converging in modulating enzyme activity.The functional significance of post-translational modifications (PTMs) to the regulation of the central carbon metabolism is the subject of this review.A genome wide analysis of PTMs indicates that several metabolic enzymes are subjected to multiple PTMs, suggesting that yeast cells can use different modifications and/or combinations of them to specifically respond to environmental changes. Glycolysis and fermentation are the pathways where phosphorylation, acetylation and ubiquitination are most frequent, while enzymes of storage carbohydrate metabolism are especially phosphorylated. Interestingly, some enzymes, such as the 6-phosphofructo-2-kinase Pfk26, the phosphofructokinases Pfk1 and Pfk2 and the pyruvate kinase Cdc19, are hubs of PTMs, thus representing central key regulation nodes. For the functionally better characterized enzymes, the role of phosphorylations and lysine modifications is discussed.This review focuses on the regulatory mechanisms of yeast carbon metabolism, highlighting the requirement of quantitative, systematical studies to better understand PTM contribution to metabolic regulation.Display Omitted
Keywords: Glycolysis; PKA; Phosphorylation; Ubiquitination; Acetylation; Saccharomyces cerevisiae;

Critical determinants of mitochondria-associated neutral sphingomyelinase (MA-nSMase) for mitochondrial localization by Vinodh Rajagopalan; Daniel Canals; Chiara Luberto; Justin Snider; Christina Voelkel-Johnson; Lina M. Obeid; Yusuf A. Hannun (628-639).
A novel murine mitochondria-associated neutral sphingomyelinase (MA-nSMase) has been recently cloned and partially characterized. The subcellular localization of the enzyme was found to be predominant in mitochondria. In this work, the determinants of mitochondrial localization and its topology were investigated.MA-nSMase mutants lacking consecutive regions and fusion proteins of GFP with truncated MA-nSMase regions were constructed and expressed in MCF-7 cells. Its localization was analyzed using confocal microscopy and sub-cellular fractionation methods. The sub-mitochondrial localization of MA-nSMase was determined using protease protection assay on isolated mitochondria.The results initially showed that a putative mitochondrial localization signal (MLS), homologous to an MLS in the zebra-fish mitochondrial SMase is not necessary for the mitochondrial localization of the murine MA-nSMase. Evidence is provided to the presence of two regions in MA-nSMase that are sufficient for mitochondrial localization: a signal sequence (amino acids 24–56) that is responsible for the mitochondrial localization and an additional ‘signal-anchor’ sequence (amino acids 77–99) that anchors the protein to the mitochondrial membrane. This protein is topologically located in the outer mitochondrial membrane where both the C and N-termini remain exposed to the cytosol.MA-nSMase is a membrane anchored protein with a MLS and a signal-anchor sequence at its N-terminal to localize it to the outer mitochondrial membrane.Mitochondrial sphingolipids have been reported to play a critical role in cellular viability. This study opens a new window to investigate their cellular functions, and to define novel therapeutic targets.
Keywords: MA-nSMase; Sphingomyelinase; Mitochondrion; MLS; Sphingolipid;

Spontaneously hyperactive MEK-Erk pathway mediates paradoxical facilitation of cell proliferation in mild hypoxia by Licht Miyamoto; Yuko Yagi; Aya Hatano; Kazuyoshi Kawazoe; Keisuke Ishizawa; Kazuo Minakuchi; Shuhei Tomita; Koichiro Tsuchiya (640-646).
Oxygen is important for common eukaryotic cells to generate ATP. Pathophysiological conditions such as ischemic diseases cause tissue hypoxia. In addition, oxygen availability in deep tissues is supposed to be far lower than surrounding atmosphere even in healthy animals, and the oxygen partial pressures in most normal tissues are estimated to be around 40–50 mmHg, so-called mild hypoxia. Recent studies have demonstrated that mild hypoxia has distinct effects on living cells from severe hypoxia. For instance, mild hypoxia was reported to promote cell reprogramming. Although severe hypoxia is known to inhibit cell proliferation, mild hypoxia has been paradoxically demonstrated to increase cell proliferation. However, it has not been clarified by which molecular mechanisms mild hypoxia evokes the discontinuous increment of cell proliferation.We established experimental conditions showing the opposite influences of mild and severe hypoxia on cell proliferation using undifferentiated Caco2 human colon carcinoma cells in order to clarify the underlying molecular mechanism.The basal activity of Erk, which is a typical mediator of mitogenic signals, is spontaneously increased specifically in cells exposed to mild hypoxia, and inhibition of MEK, an upstream kinase of the Erk, completely inhibited the mild hypoxia-induced enhancement of cell proliferation.Spontaneous hyperactivation of the MEK-Erk pathway by mild hypoxia should be the plausible molecular mechanism of the paradoxical promotion of cell proliferation.Our findings will provide clues to the molecular basis of mild hypoxia-evoked phenomena such as cell reprogramming.
Keywords: Mild hypoxia; Cell proliferation; MAP kinase cascade; Erk; Caco2 cells; Cell reprogramming;

Thermodynamic signatures of fragment binding: Validation of direct versus displacement ITC titrations by Eggert Rühmann; Michael Betz; Marie Fricke; Andreas Heine; Martina Schäfer; Gerhard Klebe (647-656).
Detailed characterization of the thermodynamic signature of weak binding fragments to proteins is essential to support the decision making process which fragments to take further for the hit-to-lead optimization.Isothermal titration calorimetry (ITC) is the method of choice to record thermodynamic data, however, weak binding ligands such as fragments require the development of meaningful and reliable measuring protocols as usually sigmoidal titration curves are hardly possible to record due to limited solubility.Fragments can be titrated either directly under low c-value conditions (no sigmoidal curve) or indirectly by use of a strong binding ligand displacing the pre-incubated weak fragment from the protein. The determination of Gibbs free energy is reliable and rather independent of the applied titration protocol.Even though the displacement method achieves higher accuracy, the obtained enthalpy–entropy profile depends on the properties of the used displacement ligand. The relative enthalpy differences across different displacement experiments reveal a constant signature and can serve as a thermodynamic fingerprint for fragments. Low c-value titrations are only reliable if the final concentration of the fragment in the sample cell exceeds 2–10 fold its KD value. Limited solubility often prevents this strategy.The present study suggests an applicable protocol to characterize the thermodynamic signature of protein–fragment binding. It shows however, that such measurements are limited by protein and fragment solubility. Deviating profiles obtained by use of different displacement ligands indicate that changes in the solvation pattern and protein dynamics most likely take influence on the resulting overall binding signature.Display Omitted
Keywords: Thermodynamic signature; Isothermal titration calorimetry; Protein–fragment binding; Displacement titrations; Low-c value titration; Thrombin;

A pore forming peptide from spider Lachesana sp. venom induced neuronal depolarization and pain by Masayoshi Okada; Gerardo Corzo; Gustavo A. Romero-Perez; Fredy Coronas; Hiroko Matsuda; Lourival D. Possani (657-666).
Arachnoid venoms contain numerous peptides with ion channel modifying and cytolytic activities.We developed a green fluorescent protein (GFP)-based assay that can monitor the changes in currents through overexpressed inwardly rectifying K+ channels (Kir2.1), in which GFP expression was increased by blockade of Kir2.1 current. Using this assay, we screened venom of many spider species. A peptide causing GFP decreasing effect was purified and sequenced. Electrophysiological and pain-inducing effects of the peptide were analyzed with whole-cell patch-clamp recordings and hot-plate test, respectively.Among venoms we screened, soluble venom from Lachesana sp. decreased the GFP expression. Purification and sequencing of the peptide showed that the peptide is identical to a pore-forming peptide purified from Lachesana tarabaevi venom. Whole cell patch-clamp recordings revealed that the peptide had no effect on Kir2.1 current. Instead, it induced a current that was attributable to the pore-formation of the peptide. The peptide was selectively incorporated into hyperpolarized, i.e., Kir2.1 expressing, cells and for this reason the peptide decreased GFP expression in our Kir2.1 assay. The pore-formation positively shifted the reversal potential and induced burst firings in the hippocampal neurons in a synaptic current-independent way. The application of the Lachesana sp. peptide induced pain-related behavior in mice.The peptide, which was found in Lachesana sp. venom, formed pores and thereby depolarized neurons and induced pain.Our data suggested an additional physiological role of the pore-forming peptides.
Keywords: Arachnoid venom; Depolarization; Inwardly rectifying K+ channel (Kir2.1); Pain; Pore-forming peptide; Thermal hyperalgesia;

Hydrophobic drug concentration affects the acoustic susceptibility of liposomes by An T. Nguyen; Peter A. Lewin; Steven P. Wrenn (667-672).
The purpose of this study was to investigate the effect of encapsulated hydrophobic drug concentration on ultrasound-mediated leakage from liposomes. Studies have shown that membrane modifications affect the acoustic susceptibility of liposomes, likely because of changes in membrane packing. An advantage of liposome as drug carrier is its ability to encapsulate drugs of different chemistries. However, incorporation of hydrophobic molecules into the bilayer may cause changes in membrane packing, thereby affecting the release kinetics. Liposomes containing calcein and varying concentrations of papaverine, a hydrophobic drug, were exposed to 20 kHz, 2.2 W cm− 2 ultrasound. Papaverine concentration was observed to affect calcein leakage although the effects varied widely based on liposome phase. For example, incorporation of 0.5 mg/mL papaverine into Ld liposomes increased the leakage of hydrophilic encapsulants by 3× within the first minute (p  = 0.004) whereas the same amount of papaverine increased leakage by only 1.5 × (p  < 0.0001). Papaverine was also encapsulated into echogenic liposomes and its concentration did not significantly affect calcein release rates, suggesting that burst release from echogenic liposomes is predictable regardless of encapsulants chemistry and concentration.
Keywords:  Controlled release; Hydrophobic drug; Liposome; Low frequency ultrasound;

Molecular recognition in naphthoquinone derivatives — G-quadruplex complexes by NMR by Benedetta Riva; Ruben Ferreira; Loana Musso; Roberto Artali; Leonardo Scaglioni; Stefania Mazzini (673-680).
G-quadruplexes have become important drug-design targets for the treatment of various human disorders such as cancer, diabetes and cardiovascular diseases. Recently, G-quadruplex structures have been visualized in the DNA of human cells and appeared to be dynamically sensitive to the cell cycle and stabilized by small molecule ligands. A small library of isoxazolo naphthoquinones (1ah), which exhibited a strong antiproliferative activity on different cancer cell lines, was studied as potential ligands of G-quadruplex DNA.The DNA binding properties of a series of the selected compounds have been analyzed by fluorescence assays. NMR/modeling studies were performed to describe the complexes between G-quadruplex DNA sequences and two selected compounds 1a and 1b. 1a and 1b in the presence of G-quadruplexes, d(T2AG3T)4, d(TAG3T2A)4 and d(T2G3T2)4, showed good ability of intercalation and the formation of complexes with 2:1 stoichiometry. 1a showed an important interaction with the sequence Pu22 belonging to the promoter of oncogenes c-myc.The ligands directly interact with the external G-tetrads of the G-quadruplexes, without alterations in the structure of the G-quadruplex core. The role of the adenine moieties over the G-tetrads in the stabilization of the complexes was discussed.The results obtained suggested that the strong antiproliferative activity of isoxazolo naphthoquinones is not due to the Hsp90 inhibition, but mainly to the interaction at the level of telomeres and/or at the level of gene promoter. These findings can be used as a basis for the rational drug design of new anticancer agents.
Keywords: G-quadruplex; NMR studies; DNA-binding drugs; Isoxazolo naphthoquinone; Molecular modeling;

Changes to both cardiac metabolism and performance accompany acute reductions in functional capillary supply by David Hauton; James Winter; Abdullah A. Al-Shammari; Eamonn A. Gaffney; Rhys D. Evans; Stuart Egginton (681-690).
The relative importance of arteriole supply or ability to switch between substrates to preserve cardiac performance is currently unclear, but may be critically important in conditions such as diabetes.Metabolism of substrates was measured before and after infusion of polystyrene microspheres in the perfused working heart to mimic random capillary loss due to microvascular disease. The effect of acute loss of functional capillary supply on palmitate and glucose metabolism together with function was quantified, and theoretical tissue oxygen distribution calculated from histological samples and ventricular VO2 estimated.Microsphere infusion led to a dose-dependent decrease in rate-pressure product (RPP) and oxygen consumption (P  < 0.001). Microsphere infusion also increased work/unit oxygen consumption of hearts (‘efficiency’) by 25% (P  < 0.01). When corrected for cardiac work palmitate oxidation remained tightly coupled to very low workloads (RPP < 2500 mm Hg/min), illustrating a high degree of metabolic control. Arteriole occlusion by microspheres decreased the density of patent capillaries (P  < 0.001) and correspondingly increased the average capillary supply area by 40% (P  < 0.01). Calculated rates of oxygen consumption declined from 16.6 ± 7.2 ml/100 ml/min to 12.4 ± 9 ml/100 ml/min following arteriole occlusion, coupled with increases in size of regions of myocardial hypoxia (Control = 22.0% vs. Microspheres = 42.2%).Cardiac mechanical performance is very sensitive to arteriolar blockade, but metabolite switching from fatty acid to glucose utilisation may also support cardiac function in regions of declining PO2.Preserving functional capillary supply may be critical for maintenance of cardiac function when metabolic flexibility is lost, as in diabetes.
Keywords: Working heart; Microspheres; Cardiac performance; Metabolism; Arteriole occlusion; Capillary domain;

Thermogenesis in white adipose tissue: An unfinished story about PPARγ by Guilherme Martins Santos; Francisco de Assis Rocha Neves; Angélica Amorim Amato (691-695).
Recruiting thermogenic adipocytes in white adipose tissue represents a potential therapeutic strategy for obesity. Interestingly, PPARγ, a major regulator of lipogenesis, is also a key factor in inducing thermogenic genes in adipose tissue.We summarize some of the recent findings regarding the biology of beige adipocytes and their potential significance for metabolic health. We also discuss the role of PPARγ in development of beige adipocyte phenotype and in inducing two apparently divergent processes, namely, lipogenesis and thermogenesis.PPARγ post-translation modifications and differential coregulator recruitment may be key factors in defining adipocyte commitment with lipogenesis or thermogenesis.Dissecting the mechanisms underlying its thermogenic effects may prompt the development of a new generation of PPARγ-based therapies.
Keywords: Beige adipocytes; Thermogenesis; PPARγ; PPARγ agonists;

A cytochrome P450 active site is buried within the protein molecule and several channels connect the catalytic cavity to the protein surface. Their role in P450 catalysis is still matter of debate. The aim of this study was to understand the possible relations existing between channels and substrate specificity.Time course studies were carried out with a collection of polycyclic substrates of increasing sizes assayed with a library of wild-type and chimeric CYP1A enzymes. This resulted in a matrix of activities sufficiently large to allow statistical analysis. Multivariate statistical tools were used to decipher the correlation between observed activity shifts and sequence segment swaps.The global kinetic behavior of CYP1A enzymes toward polycyclic substrates is significantly different depending on the size of the substrate. Mutations which are close or lining the P450 channels significantly affect this discrimination, whereas mutations distant from the P450 channels do not.Size discrimination is taking place for polycyclic substrates at the entrance of the different P450 access channels. It is thus hypothesized that channels differentiate small from large substrates in CYP1A enzymes, implying that residues located at the surface of the protein may be implied in this differential recognition.Catalysis thus occurs after a two-step recognition process, one at the surface of the protein and the second within the catalytic cavity in enzymes with a buried active site.Display Omitted
Keywords: CYP1A1; CAVER; Chimera; Polycyclic; Selectivity; Channel;

MiR-143 and rat embryo implantation by Shi Tian; Xing Su; Lu Qi; Xiao-Hua Jin; Yi Hu; Chun-Ling Wang; Xu Ma; Hong-Fei Xia (708-721).
To study the role of miR-143 during embryo implantation in rat. MiR-143 expression in rat early pregnancy was detected by Northern blot. The relation between miR-143 and Lifr predicted and confirmed by bioinformatics method, dual-luciferase activity assay, Western blot and immunohistochemistry. The role of miR-143 was detected by MTS, Edu and ranswell chamber assays.The expression level of miR-143 on gestation day 5–8 (g.d. 5–8) was higher than on g.d. 3–4 in uteri of pregnant rat. MiR-143 was mainly localized in the superficial stroma/primary decidual zone, luminal and glandular epithelia. The expression of miR-143 was not significantly influenced by pseudopregnancy, but the activation of delayed implantation and experimentally induced decidualization significantly promoted miR-143 expression. Over-expression of miR-143 in human endometrial stromal cells (ESCs) inhibited cell proliferation, migration and invasion. Knockdown of miR-143 promoted cell proliferation and invasion. The results of recombinant luciferase reporters showed that miR-143 could bind to the 3¢-untranslated region (UTR) of leukemia inhibitory factor receptor (Lifr) to inhibit Lifr translation.Uterine miR-143 may be involved in the successful pregnancy, especially during the process of blastocyst implantation through regulating Lifr.This study may have the potential to provide new insights into the understanding of miR-143 function during embryo implantation.
Keywords: miR-143; Embryo implantation; Uterus; Rat; Lifr;

Tamoxifen in combination with temozolomide induce a synergistic inhibition of PKC-pan in GBM cell lines by Joana Balça-Silva; Diana Matias; Anália do Carmo; Henrique Girão; Vivaldo Moura-Neto; Ana Bela Sarmento-Ribeiro; Maria Celeste Lopes (722-732).
Glioblastoma (GBM) is a highly proliferative, angiogenic grade IV astrocytoma that develops resistance to the alkylating agents used in chemotherapy, such as temozolomide (TMZ), which is considered the gold standard. The mean survival time for GBM patients is approximately 12 months, increasing to 14.6 months after TMZ treatment. The resistance of GBM to chemotherapy seems to be associated to genetic alterations and to the constitutive activation of several signaling pathways. Therefore, the combination of different drugs with different mechanisms of action may contribute to circumvent the chemoresistance of glioma cells. Here we describe the potential synergistic behavior of the therapeutic combination of tamoxifen (TMX), a known inhibitor of PKC, and TMZ in GBM.We used two GBM cell lines incubated in absence and presence of TMX and/or TMZ and measured cell viability, proliferation, apoptosis, cell cycle, migration ability, cytoskeletal organization and the phosphorylated amount of the p-PKC-pan.The combination of low doses of TMX with increasing doses of TMZ shows an increased antiproliferative and apoptotic effect compared to the effect with TMX alone.The combination of TMX and TMZ seems to potentiate the effect of each other. These alterations seem to be associated to a decrease in the phosphorylation status of PKC.We emphasize that TMX is an inhibitor of the p-PKC-pan and that these combination is more effective in the reduction of proliferation and in the increase of apoptosis than each drug alone, which presents a new therapeutic strategy in GBM treatment.
Keywords: Glioblastoma; Temozolomide; Tamoxifen; Chemotherapy; p-PKC; Synergism;

Many viral genomes encode a limited number of proteins, illustrating their innate efficiency in bypassing host immune surveillance. This concept of genomic efficiency is exemplified by the 9 kb RNA genome of human immunodeficiency virus 1 (HIV-1), encoding 15 proteins sub-divided according to function. The enzymatic group includes proteins such as the drug targets reverse transcriptase and protease. In contrast, the accessory proteins lack any known enzymatic or structural function, yet are essential for viral fitness and HIV-1 pathogenesis. Of these, the HIV-1 accessory protein Nef is a master manipulator of host cellular processes, ensuring efficient counterattack against the host immune response, as well as long-term evasion of immune surveillance. In particular, the ability of Nef to downmodulate major histocompatibility complex class I (MHC-I) is a key cellular event that enables HIV-1 to bypass the host's defenses by evading the adaptive immune response.In this article, we briefly review how various pathogenic viruses control cell-surface MHC-I, and then focus on the mechanisms and implications of HIV-1 Nef-mediated MHC-I downregulation via modulation of the host membrane trafficking machinery.The extensive interaction network formed between Nef and numerous membrane trafficking regulators suggests that Nef's role in evading the immune surveillance system intersects multiple host membrane trafficking pathways.Nef's ability to evade the immune surveillance system is linked to AIDS pathogenesis. Thus, a complete understanding of the molecular pathways that are subverted by Nef in order to downregulate MHC-I will enhance our understanding of HIV-1's progression to AIDS.
Keywords: AIDS; HIV-1; Nef; MHC-I; Membrane trafficking; Immune evasion;

Organic isothiocyanates (ITCs) are produced by plants, in which they are released from glucosinolates by myrosinase. ITCs are generally toxic and serve as a chemical defense against herbivorous insects and against infections by microorganisms. In mammalian tissues subtoxic concentrations of ITCs can provide protective effects against cancer and other diseases partially by induction of glutathione transferases (GSTs) and other detoxication enzymes. Thus, human consumption of edible plants rich in ITCs is presumed to provide health benefits. ITCs react with intracellular glutathione to form dithiocarbamates, catalyzed by GSTs. Formation of glutathione conjugates is central to the biotransformation of ITCs and leads to a route for their excretion. Clearly, the emergence of ITC conjugating activity in GSTs is essential from the biological and evolutionary perspective.In the present investigation an active-site-focused mutant library of GST A2-2 has been screened for enzyme variants with enhanced ITC activity.Significantly superior activities were found in 34 of the approximately 2000 mutants analyzed, and the majority of the superior GSTs featured His and Gly residues in one of the three active-site positions subjected to mutagenesis.We explored the propensity of GSTs to obtain altered substrate selectivity and moreover, identified a specific pattern of mutagenesis in GST for enhanced PEITC detoxification, which may play an important role in the evolution of adaptive responses in organisms subjected to ITCs.The facile acquisition of enhanced ITC activity demonstrates that this important detoxication function can be promoted by numerous evolutionary trajectories in sequence space.Display Omitted
Keywords: Glutathione transferase; Isothiocyanate; Directed evolution; Enzyme engineering; Detoxication;

Andrographolide (Andro) is reported to inhibit hepatoma tumor growth in our previous studies. This study aims to further search the critical signals involved in such Andro-induced inhibition.The anti-tumor effect of Andro was evaluated in vivo. Cancer PathwayFinder RT2 Profiler™ PCR array was used to find the altered genes. Real-time PCR was used to detect the mRNA expression. Protein expression was detected by Western-blot analysis, enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining. Activator protein-1 (AP-1) transcriptional activity was detected by luciferase reporter assay.Andro (10 mg/kg) inhibited hepatoma tumor growth in vivo. The expression of four genes decreased in Andro-treated tumor tissues. Among which, vascular endothelial growth factor (VEGFD) was the highest decreased gene. The decreased VEGFD expression was further confirmed by real-time PCR and immunohistochemical staining assay. Andro decreased VEGFD mRNA and protein expression in hepatoma Hep3B and HepG2 cells. Andro also decreased VEGFD amount in Hep3B cell supernatant. Andro decreased cFos protein expression and its translocation into nucleus, and also reduced AP-1 luciferase activity. Further results showed that Andro induced polyubiquitination of cFos. Proteasome inhibitor MG132 reversed the decreased expression of cFos protein, and the decreased mRNA and protein expression of VEGFD. SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), reversed the decreased expression of cFos and VEGFD induced by Andro.Andro decreased VEGFD expression in hepatoma cancer cells via inducing c-fos protein degradation, which will contribute to its anti-cancer activity, and JNK plays some roles in regulating this process.Display Omitted
Keywords: Andrographolide; Hepatoma cancer; cFos; VEGFD; Protein degradation;

Vertical ordering sensitivity of solid supported DPPC membrane to alamethicin and the related loss of cell viability by F. Domenici; F. Dell'Unto; D. Triggiani; C. Olmati; C. Castellano; F. Bordi; A. Tiezzi; A. Congiu (759-768).
Experimental studies of antimicrobial peptides interacting with lipid membranes recently attracted growing interest due to their numerous biomedical applications. However, the influence of such peptides on the structural organisation of lipid membranes in connection with the actual cell response still remains an elusive issue.X-ray diffraction was employed on detecting the sensitivity of the periodical spacing of dipalmitoyl-phosphatidyl-choline stacked as solid-supported bilayers to the presence of varying amounts of the peptide alamethicin in a wide range of peptide-to-lipid molar ratios. These results were then correlated with the effects of alamethicin on biological membranes in vitro as observed by optical microscopy and microculture tetrazolium assay on the tumour cells HeLa to provide a comprehensive and quantitative analysis of these effects, based on a dose–response relationship.The experiments allowed correlating the periodical spacing and the peptide-to-lipid molar ratio on alamethicin-dipalmitoyl-phosphatidyl-choline samples. Two different trends of periodical spacing vs. peptide-to-lipid molar ratio clearly appeared at low and high hydration levels, showing intriguing non-linear profiles. Unexpected correspondences were observed between the peptide-to-lipid molar ratio range where the changes in dipalmitoyl-phosphatidyl-choline structure occur and the alamethicin doses which alter the viability and the plasma membrane morphology of HeLa.Alamethicin might induce either mechanical or phase changes on dipalmitoyl-phosphatidyl-choline bilayers. Such easily accessible ordering information was well-calibrated to predict the alamethicin doses necessary to trigger cell death through plasma membrane alterations.This benchmark combined study may be valuable to predict bioeffects of several antimicrobial peptides of biomedical relevance.Display Omitted
Keywords: X-ray diffraction; Alamethicin; Lipid membrane; DPPC; HeLa cell; MTT assay;

Deregulation in prostaglandin (PG) biosynthesis, severe oxidative stress, inflammation and apoptosis contribute to the pathogenesis of nonsteroidal anti-inflammatory drug (NSAID)-induced gastropathy. Unfortunately, most of the prescribed anti-ulcer drugs generate various side effects. In this scenario, we could consider morin as a safe herbal potential agent against IND-gastropathy and rationalize its action systematically.Rats were pretreated with morin for 30 min followed by IND (48 mg kg− 1) administration for 4 h. The anti-ulcerogenic nature of morin was assessed by morphological and histological analysis. Its effects on the inflammatory (MPO, cytokines, adhesion molecules), ulcer-healing (COXs, PGE2), and signaling parameters (NF-κB and apoptotic signaling) were assessed by biochemical, RP-HPLC, immunoblots, IHC, RT-PCR, and ELISA at the time points of their maximal changes due to IND administration.IND induced NF-κB and apoptotic signaling in rat's gastric mucosa. These increased proinflammatory responses, but reduced the antioxidant enzymes and other protective factors. Morin reversed all the adverse effects to prevent IND-induced gastric ulceration in a PGE2 independent manner. Also, it did not affect the absorption and/or primary pharmacological activity of IND.The gastroprotective action of morin is primarily attributed to its potent antioxidant nature that also helps in controlling several IND-induced inflammatory responses.For the first time, the study reveals a mechanistic basis of morin mediated protective action against IND-induced gastropathy. As morin is a naturally abundant safe antioxidant, future detailed pharmacokinetic and pharmacodynamic studies are expected to establish it as a gastroprotective agent.
Keywords: NSAID; Morin; Gastropathy; Inflammation; Anti-inflammatory compound; Antioxidant;

Activation of muscarinic receptors in rat parotid acinar cells induces AQP5 trafficking to nuclei and apical plasma membrane by Gota Cho; Aneta M. Bragiel; Di Wang; Tomasz D. Pieczonka; Mariusz T. Skowronski; Masayuki Shono; Søren Nielsen; Yasuko Ishikawa (784-793).
The subcellular distribution of aquaporin-5 (AQP5) in rat parotid acinar cells in response to muscarinic acetylcholine receptor (mAChR) activation remains unclear.Immunoconfocal and immunoelectron microscopy were used to visualize the distribution of AQP5 in parotid acinar cells. Western blotting was used to analyze AQP5 levels in membranes. To clarify the characteristics of membrane domains associated with AQP5, detergent solubility and sucrose-density flotation experiments were performed.Under control conditions, AQP5 was diffusely distributed on the apical plasma membrane (APM) and apical plasmalemmal region and throughout the cytoplasm. Upon mAChR activation, AQP5 was predominantly located in the nucleus, APM and lateral plasma membrane (LPM). Subsequently, localization of AQP5 in the nucleus, APM and LPM was decreased. Prolonged atropine treatment inhibited mAChR agonist-induced translocation of AQP5 to the nucleus, APM and LPM. AQP5 levels were enhanced in isolated nuclei and nuclear membranes prepared from parotid tissues incubated with mAChR agonist. mAChR agonist induced AQP5 levels in both soluble and insoluble nuclear fractions solubilized with Triton X-100 or Lubrol WX. Small amounts of AQP5 in nuclei were detected using low-density sucrose gradient. When AQP5 was present in the nuclear membrane, nuclear size decreased.The activation of mAChR induced AQP5 translocation to the nucleus, APM and LPM, and AQP5 may trigger water transport across the nuclear membrane and plasma membrane in rat parotid acinar cells.AQP5 translocates to the nuclear membrane and may trigger the movement of water, inducing shrinkage of the nucleus and the start of nuclear functions.
Keywords: Aquaporin-5; Trafficking; Nuclei; Apical plasma membrane; Parotid glands; Acinar cells;

Nrf2 regulates ROS production by mitochondria and NADPH oxidase by Stjepana Kovac; Plamena R. Angelova; Kira M. Holmström; Ying Zhang; Albena T. Dinkova-Kostova; Andrey Y. Abramov (794-801).
Nuclear factor (erythroid-derived 2) factor 2 (Nrf2) is a crucial transcription factor mediating protection against oxidants. Nrf2 is negatively regulated by cytoplasmic Kelch-like ECH associated protein 1 (Keap1) thereby providing inducible antioxidant defence. Antioxidant properties of Nrf2 are thought to be mainly exerted by stimulating transcription of antioxidant proteins, whereas its effects on ROS production within the cell are uncertain.Live cell imaging and qPCR in brain hippocampal glio-neuronal cultures and explants slice cultures with graded expression of Nrf2, i.e. Nrf2-knockout (Nrf2-KO), wild-type (WT), and Keap1-knockdown (Keap1-KD).We here show that ROS production in Nrf2-KO cells and tissues is increased compared to their WT counterparts. Mitochondrial ROS production is regulated by the Keap1–Nrf2 pathway by controlling mitochondrial bioenergetics. Surprisingly, Keap1-KD cells and tissues also showed higher rates of ROS production when compared to WT, although with a smaller magnitude. Analysis of the mRNA expression levels of the two NOX isoforms implicated in brain pathology showed, that NOX2 is dramatically upregulated under conditions of Nrf2 deficiency, whereas NOX4 is upregulated when Nrf2 is constitutively activated (Keap1-KD) to a degree which paralleled the increases in ROS production.These observations suggest that the Keap1–Nrf2 pathway regulates both mitochondrial and cytosolic ROS production through NADPH oxidase.Findings supports a key role of the Keap1–Nrf2 pathway in redox homeostasis within the cell.
Keywords: ROS; Nrf2; Keap1; NADPH oxidase; NOX;

Notch signaling plays a fundamental role both in metazoan cell fate determination and in the establishment of distinct developmental cell lineages. In a yeast two-hybrid screen, we identified Chip as a binding partner of Notch. Thus, we investigated the functional significance of Notch and Chip interactions.Co-immunoprecipitation and GST pull-down experiments confirmed the physical interaction between Notch and Chip. Immunostaining revealed that Chip and Notch-intracellular domain (Notch-ICD) co-localized in cell nuclei. Loss-of-function and gain-of-function analyses of Chip were carried out using FLP/FRT and GAL4-UAS systems, respectively. Immunostaining and real-time PCR were performed to analyze the role of Chip on Notch-induced cell proliferation.Here, we report transcriptional cofactor Chip as a novel binding partner of Notch. Chip and Notch also showed strong genetic interactions, and Chip mutant clones in the dorsal compartment induced ectopic wing margins by ectopic expression of Notch and its targets, Wg and Cut. Our analyses revealed that stoichiometry of Notch and Chip is critical at the dorso-ventral (DV) boundary for wing margin formation. In addition, overexpression of Chip can rescue Notch-induced cell proliferation in larval imaginal discs.Our results indicate that Notch function in the DV boundary area is presumably dependent on Notch–Chip heterodimer formation. In addition, overexpression of Chip can rescue Notch-induced cell proliferation, presumably through titration of overexpressed Notch-ICD by excess Chip molecules. General Significance: The present study reveals that Chip is a novel interacting partner of Notch and it plays a major role in Notch-induced DV margin formation and cell proliferation.
Keywords: Drosophila; Notch; Chip; dorso-ventral boundary; cell proliferation;

Mechanisms of protection against diabetes-induced impairment of endothelium-dependent vasorelaxation by Tanshinone IIA by Yan-Hua Li; Qiang Xu; Wen-Huan Xu; Xin-Hong Guo; Shu Zhang; Yun-Dai Chen (813-823).
Impairment of endothelium-dependent vasorelaxation has been suggested to play a principle role of endothelial dysfunction in the development of cardiovascular complications of diabetes. Recent studies have demonstrated a protective effect of Tanshinone IIA (Tan) on endothelial nitric oxide synthase (eNOS)–NO pathway. However, its role in endothelium-dependent vasorelaxation in diabetes and precise mechanisms remain elusive.Sprague–Dawley rats were injected intraperitoneally with streptozotocin (STZ) to induce diabetes and then administered orally with Tan for 2 weeks. For the in vitro study, human umbilical vein endothelial cells (HUVECs) were co-incubated with Tan and high glucose for 48 h.eNOS expression and NO generation were significantly decreased in diabetic rats. These decreases were accompanied by an impairment of endothelium-dependent relaxation. Administration of Tan ameliorated the aberrant changes in eNOS expression, NO generation and endothelium-dependent relaxation in diabetic rats. Expectedly, Tan also inhibited high glucose-induced decrease of eNOS expression and NO generation in a concentration-dependent manner in HUVECs. Mechanistically, high glucose attenuated eNOS transcriptional activity through inhibiting the binding activity and nuclear translocation of Sp1 and AP-1. However, Tan did not prevent these effects. At post-transcriptional level, Tan increased eNOS expression and activity through multiple mechanisms including regulation of mRNA and protein half-life, degradation, coupling and serine 1177 phosphorylation. Rather than affecting protein phosphatase 2A (PP2A) expression and activity, Tan markedly inhibited the translocation of PP2A-A from cytosol to membrane and subsequently impaired PP2A-A/eNOS interaction, leading to prevent eNOS dephosphorylation. All these alterations underlie the protective role of Tan on eNOS expression following high glucose stimulation.Our data demonstrate that high glucose decreases eNOS expression initiating at a transcriptional level, whereas Tan prevents such effect through multiple ways of post-transcriptional mechanism.Our work provided novel mechanisms for Tan in regulating vasorelaxation and may help to better understand the cardiovascular protective action of Tan.
Keywords: Tanshinone IIA; Human umbilical vein endothelial cell; Diabetes; High glucose; NO synthase; Phosphorylation;

Andrographolide ameliorates diabetic retinopathy by inhibiting retinal angiogenesis and inflammation by Zengyang Yu; Bin Lu; Yuchen Sheng; Lingyu Zhou; Lili Ji; Zhengtao Wang (824-831).
Andrographolide (Andro) is the main compound distributed in medicinal herb Andrographis paniculata. This study aims to observe the amelioration of Andro on streptozotocin (STZ)-induced diabetic retinopathy (DR) in mice.STZ-induced non-proliferative DR (NPDR) for 2 months and proliferative DR (PDR) for 5 month in C57BL/6 mice were used in this study, respectively. Retinal vessels were observed by immunofluorescence staining for cluster of differentiation 31 (CD31). Evans blue permeation assay was used to detect the breakdown of blood-retinal barrier (BRB). Real-time PCR and immune-blot were used to detect mRNA and protein expression. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β.Retinal immunofluorescence staining with CD31 showed that Andro reduced the increased retinal vessels in STZ-induced PDR mice. Evans blue permeation results demonstrated that Andro attenuated the breakdown of BRB in STZ-induced NPDR mice. In STZ-induced PDR mice, Andro decreased the increased vascular endothelial growth factor (VEGF) in serum and vitreous cavity, and reduced the increased retinal mRNA expression of VEGF and its receptors. In STZ-induced NPDR mice, Andro abrogated the nuclear translocation of nuclear factor κB (NF-κB) p65 and early growth response-1 (Egr-1), and reduced the increased phospho-NF-κBp65, -inhibitor of kappa B (IκB), and -IκB kinase (IKK). Andro also decreased the increased serum and retinal mRNA expression of TNF-α, IL-6, IL-1β, serpine1, and tissue factor (TF).Andro ameliorates DR via attenuating retinal angiogenesis and inflammation, and VEGF, NF-κB, and Egr1 signaling pathways all play important roles in this process.Display Omitted
Keywords: Andrographolide; Diabetic retinopathy; Angiogenesis; Inflammation;

The Nav1.2 channel is regulated by GSK3 by Thomas F. James; Miroslav N. Nenov; Norelle C. Wildburger; Cheryl F. Lichti; Jonathan Luisi; Fernanda Vergara; Neli I. Panova-Electronova; Carol L. Nilsson; Jai S. Rudra; Thomas A. Green; Demetrio Labate; Fernanda Laezza (832-844).
Phosphorylation plays an essential role in regulating voltage-gated sodium (Nav) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Nav channels. We posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Nav channels.We used patch-clamp electrophysiology to record sodium currents from Nav1.2 channels stably expressed in HEK-293 cells. mRNA and protein levels were quantified with RT-PCR, Western blot, or confocal microscopy, and in vitro phosphorylation and mass spectrometry to identify phosphorylated residues.We found that exposure of cells to GSK3 inhibitor XIII significantly potentiates the peak current density of Nav1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3β suppressed Nav1.2-encoded currents. Neither mRNA nor total protein expression was changed upon GSK3 inhibition. Cell surface labeling of CD4-chimeric constructs expressing intracellular domains of the Nav1.2 channel indicates that cell surface expression of CD4-Nav1.2 C-tail was up-regulated upon pharmacological inhibition of GSK3, resulting in an increase of surface puncta at the plasma membrane. Finally, using in vitro phosphorylation in combination with high resolution mass spectrometry, we further demonstrate that GSK3β phosphorylates T1966 at the C-terminal tail of Nav1.2.These findings provide evidence for a new mechanism by which GSK3 modulates Nav channel function via its C-terminal tail.These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.
Keywords: Sodium channel; Patch-clamp electrophysiology; Glycogen synthase kinase 3; Confocal microscopy; Protein–protein interactions;

A simple, fast and cost-effective method of synthesis of cupric oxide nanoparticle with promising antibacterial potency: Unraveling the biological and chemical modes of action by Ruchira Chakraborty; Raj Kumar Sarkar; Arijit Kumar Chatterjee; Unnikrishnan Manju; Asoke Prasun Chattopadhyay; Tarakdas Basu (845-856).
Gradual attainment of bacterial resistance to antibiotics led us to develop a robust method of synthesis of stable, colloidal cupric oxide nanoparticle of physiological pH with potential antibacterial action.Cu(II) oxide NP was synthesized by reduction–oxidation of CuCl2, using polyvinyl alcohol as stabilizer. Characteristics and antibacterial activity of the particles were investigated by techniques like UV–Vis spectrophotometry, DLS, AFM, TEM, EDS, FTIR, AAS, agar plating, FACS, gel electrophoresis and XPS.The NPs were about 50 nm in size and cubic in shape with two surface plasmon peaks at 266 and 370 nm and had semi-conducting behavior with a band gap of 3.40 and 3.96 eV. About 80% of precursor CuCl2 was converted to NP. The minimum inhibitory and the minimum bactericidal concentrations of CuO-NP were respectively 120 and 160 μg/mL for Escherichia coli and 180 and 195 μg/mL for Staphylococcus aureus in Luria–Bertani medium. In growth media, the NPs got modified by media organics with displacement of the stabilizer PVA molecules. This modified NP (around 240 nm) killed cells by generating ROS, which finally caused membrane lipid per-oxidation and chromosomal DNA degradation in NP-treated cells.Reports indicate that we are among the few who had prepared CuO-NP in colloidal form. The antibacterial potency of our particle in growth media was much promising than other reports. Our findings demonstrated that ‘particle-specific’ effect, not ‘ion-specific’ one, was responsible for the NP action.The NP may be used as a sterilizing agent in various bioprocesses and as substituent of antibiotics, after thorough toxicological study.
Keywords: Cupric oxide nanoparticle; Colloidal suspension; Antibacterial action; ROS generation; Modification of Cu(II) oxide NP; Growth media organics;