Check out our New Publishers' Select for Free Articles

BBA - General Subjects (v.1810, #4)

Editorial Board (pp. i).

Purification, biochemical characterization and antifungal activity of a new lipid transfer protein (LTP) from Coffea canephora seeds with α-amylase inhibitor properties by Umberto Zottich; Maura Da Cunha; André O. Carvalho; Germana B. Dias; Nádia C.M. Silva; Izabela S. Santos; Viviane V. do Nacimento; Emílio C. Miguel; Olga L.T. Machado; Valdirene M. Gomes (pp. 375-383).

A growing number of cysteine-rich antimicrobial peptides (AMPs) have been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides, which include lipid transfer proteins (LTPs), play an important role in the protection of plants against microbial infection.Peptides from Coffea canephora seeds were extracted in Tris–HCl buffer (pH 8.0), and chromatographic purification of LTP was performed by DEAE and reverse-phase HPLC. The purified peptide was submitted to amino acid sequence, antimicrobial activity and mammalian α-amylase inhibitory analyses.The purified peptide of 9kDa had homology to LTPs isolated from different plants. Bidimensional electrophoresis of the 9kDa band showed the presence of two isoforms with pIs of 8.0 and 8.5. Cc-LTP₁ exhibited strong antifungal activity, against Candida albicans, and also promoted morphological changes including the formation of pseudohyphae on Candida tropicalis, as revealed by electron micrograph. Our results show that Cc-LTP₁ interfered in a dose-dependent manner with glucose-stimulated, H⁺-ATPase-dependent acidification of yeast medium and that the peptide permeabilized yeast plasma membranes to the dye SYTOX green, as verified by fluorescence microscopy. Interestingly, we also showed for the first time that the well characterized LTP₁ family, represented here by Cc-LTP₁, was also able to inhibit mammalian α-amylase activity in vitro.In this work we purified, characterized and evaluated the in vitro effect on yeast of a new peptide from coffee, named Cc-LPT1, which we also showed, for the first time, the ability to inhibit mammalian α-amylase activity.► Cc-LTP₁ (9kDa band) showed the presence of two isoforms with pIs of 8.0 and 8.5. ► Cc-LTP₁ exhibited strong antifungal activity against Candida albicans yeast. ► Cc-LTP₁ interfered with glucose-stimulated H⁺-ATPase-dependent acidification of yeast. ► Cc-LTP₁ permeabilized yeast plasma membranes to the dye SYTOX green. ► Cc-LTP₁, was also able to inhibit mammalian α-amylase activity in vitro.

Keywords: Antimicrobial peptides; Lipid transfer protein; α-Amylase inhibitor; Pathogenic yeast; Candida albicans

Ergosta-4,6,8(14),22-tetraen-3-one induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells by Ying-Yong Zhao; Xuefeng Shen; Xu Chao; Charlene C. Ho; Xian-Long Cheng; Yongmin Zhang; Rui-Chao Lin; Ke-Jun Du; Wen-Jing Luo; Jing-Yuan Chen; Wen-Ji Sun (pp. 384-390).

Mushrooms have been used in Asia as traditional foods and medicines for a long time. Ergosta-4,6,8(14),22-tetraen-3-one (ergone) is one of the well-known bioactive steroids, which exists widely in various medicinal fungi such as Polyporus umbellatus, Russula cyanoxantha, and Cordyceps sinensis. Ergone has been demonstrated to possess cytotoxic activity. However, the molecular mechanisms by which ergone exerts its cytotoxic activity are currently unknown.In the present study, ergone possessed a remarkable anti-proliferative activity toward human hepatocellular carcinoma HepG2 cells. We assayed the cell cycle by flow cytometry using PI staining; investigated the exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane by the FITC-annexin V/PI staining; observed the nuclear fragmentation by Hoechst 33258 staining and studied the protein expression of Bax, Bcl-2, p-53, procaspase-3, -8, -9, PARP and cleaved PARP by Western blotting analysis.Cells treated with ergone showed typical markers of apoptosis: G2/M cell cycle arrest, chromatin condensation, nuclear fragmentation, and phosphatidylserine exposure. Furthermore, PARP-cleavage; activation of caspase-3, -8, -9; up-regulation of Bax and down-regulation of Bcl-2 were observed in HepG2 cells treated with ergone, which show that both the intrinsic and extrinsic apoptotic pathways are involved in ergone-induced apoptosis in HepG2 cells. Ergosta-4,6,8(14),22-tetraen-3-one induces G2/M cell cycle arrest and apoptosis in HepG2 cells in a caspase-dependent manner.In this study, we reported for the first time that ergone-induced apoptosis through activating the caspase. These results would be useful for the further utilization of many medicinal fungi in cancer treatment.

Keywords: Medicinal fungi; Polyporus umbellatus; Ergosta-4,6,8(14),22-tetraen-3-one; G2/M phase arrest; Apoptosis; Human hepatocellular carcinoma HepG2 cell

Redox state of Troponin C Cysteine in the D/E helix alters the C-domain affinity for the thin filament of vertebrate striated muscle by José Renato Pinto; Valeria Pereira de Sousa; Martha M. Sorenson (pp. 391-397).

Despite a broad spectrum of structural studies, it is not yet clear whether the D/E helix of troponin C (TnC) contributes to the interaction of TnC with troponin I (TnI). Redox modifications at Cys 98 in the D/E helix were explored for clues to TnC binding to the thin filament off-state, using recombinant wild-type TnC and an engineered mutant without Cys (Cys98Leu).Recombinant proteins and rabbit psoas skinned fibres were reduced with dithiothreitol (DTT) and variously recombined. Changes in affinity of reduced or oxidised TnC for the thin filament were evaluated via TnC binding and dissociation, using a standardized test for maximal force as an index of fibre TnC content.All oxidation and reduction effects observed were reversible and led to changes in TnC content. Oxidation (H₂O₂) reduced TnC affinity for the filament; reduction (DTT) increased it. Reducing other fibre proteins had no effect. Binding of the Cys-less TnC mutant was not altered by DTT, nor was dissociation of wild-type TnC from reconstituted hybrids (skeletal TnC in cardiac trabeculae). Thus when Cys 98 in the D/E helix of TnC is fully reduced, its binding affinity for the thin filament of skeletal muscle is enhanced and helps to anchor it to the filament.Signal transmission between TnC and the other proteins of the regulatory complex is sensitive to the redox state of Cys 98.► TnC affinity for the thin filament is sensitive to the redox state. ► DTT prevents TnC dissociation from the thin filament. ► H₂O₂ increases TnC dissociation from the thin filament. ► DTT does not affect TnC-C98L dissociation from the thin filament. ► Cys 98 in the D/E helix of TnC helps to anchor it to the thin filament.

Keywords: Abbreviations; P; _o; maximum Ca; ²⁺; -activated tension in native skinned fibre; P; _recon; maximum Ca; ²⁺; -activated tension following reconstitution with exogenous TnC; TFP; trifluoperazine; Tn; troponin; TnC; troponin C; rTnC; recombinant TnC; sTnC; native skeletal muscle TnC; TnI; troponin I; TnT; troponin T; Ip; inhibitory peptide of TnITroponin I-C affinity; D/E helix; Cysteine 98; Redox state; Skinned fibres; Rat trabeculae

Differences in the fractional abundances of carbohydrates of natural and recombinant human tissue factor by Jolanta Krudysz-Amblo; Mark E. Jennings II; Dwight E. Matthews; Kenneth G. Mann; Saulius Butenas (pp. 398-405).

Tissue factor (TF) is a single polypeptide integral membrane glycoprotein composed of 263 residues and is essential to life in its role as the initiator of blood coagulation. Previously we have shown that the activity of the natural placental TF (pTF) and the recombinant TF (rTF) from Sf9 insect cells is different (Krudysz-Amblo, J. et al (2010) J. Biol. Chem. 285, 3371–3382).In this study, using mass spectrometry, we show by quantitative analysis that the extent of glycosylation varies on each protein.Fractional abundance of each glycan composition at each of the three glycosylation sites reveals the most pronounced difference to be at asparagine (Asn) 11. This residue is located in the region of extensive TF-factor VIIa (FVIIa) interaction. Carbohydrate fractional abundance at Asn11 revealed that glycosylation in the natural placental TF is much more prevalent (~76%) than in the recombinant protein (~20%). The extent of glycosylation on Asn124 and Asn137 is similar in the two proteins, despite the pronounced differences in the carbohydrate composition. Additionally, 77% of rTF exists as TF des-1, 2 (missing the first two amino acids from the N-terminus). In contrast, only 31% of pTF is found in the des-1, 2 form.These observations may attribute to the difference in the ability of TF–FVIIa complex to activate factor X (FX).Structural and functional comparison of the recombinant and natural protein advances our understanding and knowledge on the biological activity of TF.► Natural human placental TF exhibits enhanced activity relative to a recombinant form. ► Major differences in carbohydrates and extent of glycosylation between the two forms. ► Extent and glycosylation type may augment TF activity in various systems/tissues. ► New insights into the biological contribution of carbohydrates on TF activity.

Keywords: Mass spectrometry; Fractional abundance; Carbohydrates; Tissue factor

Demonstration of the hepatocyte growth factor signaling pathway in the in vitro neuritogenic activity of chondroitin sulfate from ray fish cartilage by Taishi Hashiguchi; Takanari Kobayashi; Duriya Fongmoon; Ajaya Kumar Shetty; Shuji Mizumoto; Nobuyuki Miyamoto; Toshikazu Nakamura; Shuhei Yamada; Kazuyuki Sugahara (pp. 406-413).

Chondroitin sulfate (CS) is a ubiquitous component of the cell surface and extracellular matrix and its sugar backbone consists of repeating disaccharide units: D-glucuronic acid (GlcUA)β1-3 N-acetyl-D-galactosamine (GalNAc). Although CS participates in diverse biological processes such as growth factor signaling and the nervous system's development, the mechanism underlying the functions is not well understood.CS was isolated from ray fish cartilage, an industrial waste, and its structure and neurite outgrowth-promoting (NOP) activity were analyzed to investigate a potential application to nerve regeneration.The major disaccharide unit in the CS preparation was GlcUA-GalNAc(6- O-sulfate) (61.9%). Minor proportions of GlcUA-GalNAc(4- O-sulfate) (27.0%), GlcUA(2- O-sulfate)-GalNAc(6- O-sulfate) (8.5%), and GlcUA-GalNAc (2.7%) were also detected. The preparation showed NOP activity in vitro, and this activity was suppressed by antibodies against hepatocyte growth factor (HGF) and its receptor c-Met, suggesting the involvement of the HGF signaling pathway in the expression of the in vitro NOP activity of the CS preparation. The specific binding of HGF to the CS preparation was also demonstrated by surface plasmon resonance spectroscopy.The NOP activity of CS from ray cartilage was demonstrated to be expressed through the HGF signaling pathway, suggesting that ray cartilage CS may be useful for studying the cooperative function of CS and HGF.► Chondroitin sulfate (CS) was isolated from the cartilage of ray fish. ► The structure and neurite outgrowth-promoting (NOP) activity were characterized. ► The NOP activity was suppressed by an anti-hepatocyte growth factor (HGF) antibody. ► The specific binding of HGF to the CS preparation was demonstrated. ► The HGF signaling seems to be involved in the NOP activity of the CS preparation.

Keywords: Abbreviations; GAG; glycosaminoglycan; CS; chondroitin sulfate; GlcUA; D-glucuronic acid; IdoUA; L-Iduronic acid; GalNAc; N; -acetyl-D-galactosamine; PTN; pleiotrophin; MK; midkine; HGF; hepatocyte growth factor; GDNF; glial cell line-derived neurotrophic factor; P-ORN; poly-DL-ornithine; HPLC; high-performance liquid chromatography; CSase; chondroitinase; NOP; neurite outgrowth-promoting; 2AB; 2-aminobenzamide; ∆HexUA; 4-deoxy-L-; threo; -hex-4-enepyranosyluronic acid; IU; international unitChondroitin sulfate; Cartilage; Neurite outgrowth-promoting activity; Hepatocyte growth factor; c-Met receptor

A novel retinoic acid analogue, 7-hydroxy retinoic acid, isolated from cyanobacteria by Kunimitsu Kaya; Fujio Shiraishi; Hideaki Uchida; Tomoharu Sano (pp. 414-419).

All- trans retinoic acid (RA) is a low-molecular compound derived from vitamin A. It induces events in various ways by binding with the retinoic acid receptor (RAR), a nuclear receptor, in animal cells. RA and its metabolites have been found in animal tissues. In this paper, we report a novel RA analogue found in cyanobacterial cells, describe the method for its isolation, and compare its photo-stability with that of all- trans RA.The new A analogue was extracted from cells of Microcystis aeruginosa and Spirulina sp. and fractionated by high-performance liquid chromatography. The analogue was analysed using a yeast two-hybrid assay method to measure in vitro RAR-agonistic activity. Liquid chromatography–mass spectrometry/mass spectrometry analyses was performed to elucidate the chemical structure of this RA analogue.The results of the analysis of the fragments revealed that the novel RA analogue was 7-hydroxy RA. The yields from 3.5μg (4.5% of the total RAR-agonistic activity of Spirulina sp. cells) of 7-hydroxy RA was a mixture of 4 isomers due to cis–trans isomerisation coupled with keto–enol tautomerism; its relative RAR agonistic activity was 0.49±0.01 (n=3) when the activity of all trans RA was set up to 1.00. Under fluorescent light, the mixture of 7-hydroxy RA isomers was more stable than all- trans RA.We isolated a novel RAR-activating compound, 7-hydroxy RA, from cyanobacteria.7-hydroxy RA is more stable than all- trans RA under UV-A.► A novel RAR-activating compound, 7-hydroxy retinoic acid (RA), was isolated from cyanobacteria. ► 7-Hydroxy RA is consisted of 4 isomers due to cis-trans isomerization coupled with keto-enol tautomerism. ► 7-Hydroxy RA is more stable than all-trans RA under UV-A.

Keywords: 7-hydroxy retinoic acid; RAR agonistic activity; Cyanobacteria; UV stability

Molecular characterization of an insecticide-induced novel glutathione transferase in silkworm by Kohji Yamamoto; Hirofumi Ichinose; Yoichi Aso; Yutaka Banno; Makoto Kimura; Takashi Nakashima (pp. 420-426).

The glutathione transferase (GST) superfamily is involved in the detoxification of various xenobiotics. We have identified a GST mRNA that was induced in the fat bodies of a silkworm strain exhibiting diazinon resistance and have investigated the enzyme properties of this GST.A soluble recombinant protein was overexpressed in Escherichia coli. Amino acid residues of interest were changed to alanine by site-directed mutagenesis.Phylogenetic analysis of the deduced amino acid sequence indicates that this GST belongs to an unclassified group previously reported in mosquitoes. This enzyme, named bmGSTu, has highly conserved amino acid residues, including Tyr7, Ser12 and Asn50. A recombinant bmGSTu was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic analysis of bmGSTu mutants indicated that Tyr7, Ser12 and Asn50 are involved in enzyme function.These results support the hypothesis that bmGSTu may play a role in insecticide resistance in Bombyx mori.► We have identified an insecticide-induced GST in a silkworm. ► Phylogenetic analysis indicates that this GST belongs to an unclassified GST. ► bmGSTu catalyzed the biotranslation of GSH with a synthetic substrate of GST. ► bmGSTu mutants indicated that Tyr7, Ser12 and Asn50 are involved in the catalysis.

Keywords: Abbreviations; CBB; Coomassie Brilliant Blue R250; CDNB; 1-chloro-2,4-dinitrobenzene; ECA; ethacrynic acid; GSH; glutathione; GST; glutathione transferase; 4-HNE; 4-hydroxynonenal; IPTG; isopropyl 1-thio-β-; d; -galactoside; 4-NPA; 4-nitrophenyl acetate; LB; Luria–Bertani medium; PCR; polymerase chain reaction; RT-PCR; reverse transcriptase PCR; SDS-PAGE; polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate Bombyx mori; Glutathione; Glutathione transferase; Lepidoptera; Site-directed mutagenesis

FRET analysis of protein tyrosine kinase c-Src activation mediated via aryl hydrocarbon receptor by Bin Dong; Wei Cheng; Wen Li; Jie Zheng; Dalei Wu; Fumio Matsumura; Christoph Franz Adam Vogel (pp. 427-431).

Activation of the protein tyrosine kinase c-Src (c-Src kinase) induced by the exposure to the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown in various cell types. Most previous works used Western blot analysis to detect the phosphorylation on the Tyr416 residue, which activates c-Src kinase.Here we compared the results of c-Src tyrosine phosphorylation via aryl hydrocarbon receptor (AhR)-dependent mechanisms from Western blot analysis with fluorescent resonance energy transfer (FRET) assay detecting c-Src activation after treatment with TCDD to activate AhR in two different human cell types.Western blot analyses show time-dependent phosphorylation of c-Src by TCDD in HepG2 and MCF-10A cells. Data from FRET assay visualized and quantified the activation of c-Src kinase induced by TCDD in living cells of both cell types. The FRET efficiency decreased by 20%, 5min after TCDD treatment and continued decreasing until the end of the experiment, 25min after TCDD treatment. PP2, a c-Src specific inhibitor, suppressed both TCDD- and epidermal growth factor- (EGF) induced c-Src activation. In contrast, the AhR antagonist 3′-methoxy-4′nitroflavone (MNF) blocked only TCDD- but not EGF-induced activation of c-Src.The current study shows that the early activation of c-Src via EGF and AhR signaling pathways can be visualized in living cells using the FRET assay which is in line with Western blot analysis.The FRET assay provides a useful tool to visualize and quantify c-Src kinase activation via AhR in living cells.► Activation of c-Src kinase by TCDD in living cells. ► FRET assay to detect early and AhR-dependent activation of c-Src kinase. ► Regulatory role of AhR and cPLA2 during activation of c-Src in living cells.

Keywords: AhR; COX-2; c-Src; EGF; FRET; TCDD

Cell cycle-dependent conjugation of endogenous BRCA1 protein with SUMO-2/3 by Aurélie Vialter; Anne Vincent; Aïcha Demidem; Daniel Morvan; Georges Stepien; Nicole Dalla Venezia; Pascale G. Rio (pp. 432-438).

BRCA1, the main breast and ovarian cancer susceptibility gene, has a key role in maintenance of genome stability, cell cycle and transcription regulation. Interestingly, some of the numerous proteins which interact with BRCA1 protein undergo conjugation with small ubiquitin-like modifiers (SUMO). This post-translational modification is related to transcription, DNA repair, nuclear transport, signal transduction, and to cell cycle stress response.Protein sequence analysis suggests that sumoylation target sites belong to the RING finger and BRCT domains (BRCA1 C-terminus), two crucial regions for BRCA1 function. Moreover putative SUMO interacting motifs are present in the sequence of many proteins of BRCA1 network. Using immunoprecipitations and western blotting, we show the conjugation of endogenous nuclear BRCA1 protein with SUMO-2/3. BRCA1 conjugation with SUMO-2/3 is linked to the cell cycle in a cell line dependent manner since no cell cycle dependence of sumoylation is observed in MCF7 breast cancer cells. In contrast, BRCA1 conjugation with SUMO-2/3 is linked to the oxidative stress independently to the cell line, in DU145, MCF7 and 293 T cells.Our data reveal a new BRCA1 regulation pathway implying sumoylation in response to cell cycle progression and oxidative stress, providing a possible mechanism for the involvement of BRCA1 gene in tumorigenesis.► Nuclear BRCA1 protein is conjugated with SUMO-2/3. ► BRCA1 sumoylation is linked to the cell cycle in a cell line dependent manner. ► BRCA1 sumoylation is maximum in G1. ► BRCA1 sumoylation is linked to the oxidative stress. ► BRCA1-associated proteins are sumoylated and/or contain potential SUMO-binding motifs.

Keywords: Abbreviations; SUMO; small ubiquitin-like modifiers; SIM; SUMO interacting motifs; BRCT; BRCA1 C-terminusBRCA1; SUMO-2/3; Cell cycle; Protein processing; Post-translational; Oxidative stress

A novel GRAIL E3 ubiquitin ligase promotes environmental salinity tolerance in euryhaline tilapia by Diego F. Fiol; Enio Sanmarti; Andreana H. Lim; Kultz Dietmar Kültz (pp. 439-445).

Tilapia ( Oreochromis mossambicus) are euryhaline fishes capable of tolerating large salinity changes. In a previous study aimed to identify genes involved in osmotolerance, we isolated an mRNA sequence with similarity to GRAIL (Gene Related to Anergy In Lymphocytes), which is a critical regulator of adaptive immunity and development. Tilapia GRAIL contains a PA (protease associated) domain and a C3H2C3 RING finger domain indicative of E3 ubiquitin ligase activity.Western blots analysis was used to assess GRAIL expression pattern and responses to hyperosmotic stress. Immunohistochemistry was used to reveal the cellular localization of GRAIL in gill epithelium. Overexpression in HEK293 T-Rex cells was used to functionally characterize tilapia GRAIL. Salinity stress causes strong up-regulation of both mRNA and protein levels of tilapia GRAIL in gill epithelium. Tissue distribution of GRAIL protein is mainly confined to gill epithelium, which is the primary tissue responsible for osmoregulation of teleost fishes. Overexpression of tilapia GRAIL in HEK293 cells increases cell survival (cell viability) while decreases apoptosis during salinity challenge.Our data indicate that tilapia GRAIL is a novel E3 ubiquitin ligase involved in osmotic stress signaling, which promotes environmental salinity tolerance by supporting gill cell function during hyperosmotic stress.Involvement of tilapia GRAIL in the osmotic stress response suggests that GRAIL E3 ubiquitin ligases play a broader role in environmental stress responses, beyond their documented functions in adaptive immunity and development.Display Omitted► Salinity stress induces Tilapia GRAIL mRNA and protein in gill epithelium. ► Tilapia GRAIL is highly expressed in gills and weakly in other tissues. ► Tilapia GRAIL is localized at the very tip of gill secondary lamellae. ► Tilapia GRAIL promotes cell survival in hyperosmotic media. ► Tilapia GRAIL suppresses programmed cell death (apoptosis) in hyperosmotic media.

Keywords: Hyperosmotic stress; Oreochromis mossambicus; GRAIL; GREUL; Goliath

Redox regulation of ERK1/2 activation induced by sphingosine 1-phosphate in fibroblasts: Involvement of NADPH oxidase and platelet-derived growth factor receptor by Serena Catarzi; Cecilia Romagnoli; Gemma Marcucci; Fabio Favilli; Teresa Iantomasi; Maria T. Vincenzini (pp. 446-456).

Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite synthesized after stimulation with growth factors or cytokines. S1P extracellular effects are mediated through specific Gi-protein coupled receptors (GPCRs). Recently, we demonstrated in NIH3T3 fibroblasts stimulated by platelet-derived growth factor (PDGF) or S1P the NADPH oxidase activation and the H₂O₂ intracellular level increase trough the Gi protein involvement.NIH3T3 fibroblast cell cultures were used. Western blot and quantitative analyses by Chemidoc-Quantity-One software were performed. H₂O₂ level was assayed by fluorescence spectrophotometric analysis, and cell proliferation by counted manually or ELISA kit.This study demonstrates, in NIH 3T3 fibroblasts, a novel redox regulated mechanism of S1P-induced activation of ERK 1/2 related to NADPH oxidase activity and intracellular H₂O₂ level increase with PDGF receptor tyrosine kinase involvement through a transactivation mechanism. This event is mediated by S1P₁ and S1P₃ receptors by Gi proteins and can contribute to S1P mitogenic signaling.These results can be related to mechanisms of cross-talk previously identified between receptor tyrosine kinase, including PDGFreceptor, and several GPCR ligands.The redox-sensitive ERK1/2 and PDGFr tyrosine kinase activity could be targets for therapies in diseases in which deregulation of intracellular oxidative status and the consequent alteration of S1P and/or PDGF signaling pathway are involved.► S1P induces activation of ERK1/2 by H₂O₂ production. ► Involvement of tyrosine kinase of PDGFr on redox regulation of ERK1/2. ► Cross-talk by transactivation between receptors of S1P and PDGFr.

Keywords: Sphingosine 1-Phosphate; ERK1/2 Kinase; NADPH oxidase; PDGFr tyrosine kinase, Receptor cross-talk

A novel vanadium transporter of the Nramp family expressed at the vacuole of vanadium-accumulating cells of the ascidian Ascidia sydneiensis samea by Tatsuya Ueki; Nobuaki Furuno; Hitoshi Michibata (pp. 457-464).

Vanadium is an essential transition metal in biological systems. Several key proteins related to vanadium accumulation and its physiological function have been isolated, but no vanadium ion transporter has yet been identified.We identified and cloned a member of the Nramp/DCT family of membrane metal transporters ( AsNramp) from the ascidian Ascidia sydneiensis samea, which can accumulate extremely high levels of vanadium in the vacuoles of a type of blood cell called signet ring cells (also called vanadocytes). We performed immunological and biochemical experiments to examine its expression and transport function.Western blotting analysis showed that AsNramp was localized at the vacuolar membrane of vanadocytes. Using the Xenopus oocyte expression system, we showed that AsNramp transported VO²⁺ into the oocyte as pH-dependent manner above pH 6, while no significant activity was observed below pH 6. Kinetic parameters ( K_m and V_max) of AsNramp-mediated VO²⁺ transport at pH 8.5 were 90nM and 9.1pmol/oocyte/h, respectively. A rat homolog, DCT1, did not transport VO²⁺ under the same conditions. Excess Fe²⁺, Cu²⁺, Mn²⁺, or Zn²⁺ inhibited the transport of VO²⁺. AsNramp was revealed to be a novel VO²⁺/H⁺ antiporter, and we propose that AsNramp mediates vanadium accumulation coupled with the electrochemical gradient generated by vacuolar H⁺-ATPase in vanadocytes.This is the first report of identification and functional analysis on a membrane transporter for vanadium ions.► A homolog of Nramp, AsNramp, was cloned from a vanadium-rich ascidian. ► AsNramp is a novel VO²⁺/H⁺ antiporter on the vacuolar membrane. ► A rat homolog, DCT1, did not transport VO²⁺ under the same conditions. ► This is the first report on a membrane transporter for vanadium ions.

Keywords: Membrane protein; Transport metal; Vanadium; Ascidian

Structure-activity relationship of acridine derivatives to amyloid aggregation of lysozyme by Andrea Antosova; Beatrice Chelli; Eva Bystrenova; Katarina Siposova; Francesco Valle; Jan Imrich; Maria Vilkova; Pavol Kristian; Fabio Biscarini; Zuzana Gazova (pp. 465-474).

Amyloid-related diseases (such as Alzheimer's disease or diabetes type II) are associated with self-assembly of protein into amyloid aggregates.Spectroscopic and atomic force microscopy were used to determine the ability of acridines to affect amyloid aggregation of lysozyme.We have studied the effect of acridine derivatives on the amyloid aggregation of lysozyme to investigate the acridine structure-activity relationship. The activity of the effective planar acridines was characterized by the half-maximum depolymerization concentration DC₅₀ and half-maximal inhibition concentration IC₅₀. For the most effective acridine derivatives we examined their interaction with DNA and their effect on cell viability in order to investigate their eventual influence on cells. We thus identified planar acridine derivatives with intensive anti-amyloid activity (IC₅₀ and DC₅₀ values in micromolar range), low cytotoxicity and weak ability to interfere with the processes in the cell.Our findings indicate that both the planarity and the tautomerism of the 9-aminoacridine core together with the reactive nucleophilic thiosemicarbazide substitution play an important role in the anti-amyloid activities of studied derivatives.The present findings favor the application of the selected active planar acridines in the treatment of amyloid-related diseases.Display Omitted► Acridine derivatives affect amyloid aggregation of lysozyme. ► Extent of amyloid inhibition and depolymerization depends on acridine structure. ► Planarity and tautomerism of 9-aminoacridine core enhance anti-amyloid activity. ► IC50 and DC50 for most effective planar acridines at low micromolar concentrations. ► Identification of acridines with high antiamyloid activity and low cytotoxicity.

Keywords: Protein amyloid aggregation; Lysozyme; Acridine; Atomic force microscopy; Cytotoxicity

Hypertonicity-enhanced TNF-α release from activated human monocytic THP-1 cells requires ERK activation by Yung-Chen Chou; Joen-Rong Sheu; Chi-Li Chung; Che-Jen Hsiao; Po-Jen Hsueh; George Hsiao (pp. 475-484).

Hypertonic stress enhances tumor necrosis factor (TNF)-α expression in activated monocytes. However, the underlying mechanism is unknown. The produced TNF-α is primarily cleaved and released by TNF-α-converting enzyme (TACE), and the surface expression of TACE is down-regulated by endocytosis. As hypertonicity inhibits endocytosis, we evaluated the mechanism of hypertonicity-induced TNF-α release from activated human monocytic THP-1 cells.THP-1 cells were stimulated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) in the presence or absence of hypertonic agents (150mM sucrose or 150–300mM NaCl). The amount of TNF-α mRNA and protein, surface expression of TACE and activation of signaling pathways (mitogen-activated protein kinase, Akt and NF-κB) were assayed.Hypertonic sucrose and NaCl significantly enhanced TNF-α release from THP-1 cells upon LPS or PMA stimulation. Hypertonic sucrose and other endocytosis inhibitors increased surface expression of TACE, but their effects on TNF-α release were inconsistent. This enhancement effect by hypertonicity was not attenuated by inhibition of TACE or IκB kinase, but it was blocked by cycloheximide and a MAP/ERK kinase inhibitor. The LPS- or PMA-induced TNF-α mRNA expression was not increased; rather, it was inhibited by hypertonicity. ERK1/2 was re-activated after sucrose treatment in LPS-stimulated THP-1 cells.Hypertonicity-enhanced TNF-α protein synthesis from LPS- or PMA-activated THP-1 cells requires ERK activation and may proceed without TACE.A vast amount of TNF-α production was regulated by a crucial post-transcriptional manner in activated human monocytic leukemia cells, and it may possibly be contributed to the cachexia condition.►ERK activation is required for hypertonicity-enhanced TNF-α production. ►This enhancement occurred through an NF-κB-independent pathway. ►It could be independent of endocytosis, TACE activation and TNF-α transcription.

Keywords: Hypertonicity; TNF-α; LPS; PMA; Endocytosis; TACE

Interaction of berberine, palmatine, coralyne, and sanguinarine to quadruplex DNA: A comparative spectroscopic and calorimetric study by Kakali Bhadra; Gopinatha Suresh Kumar (pp. 485-496).

Interaction of isoquinoline alkaloids berberine, palmatine, coralyne and sanguinarine with human telomeric quadruplex DNA, dAGGG(TTAGGG)₃, has been investigated and compared with ethidium.Biophysical techniques such as absorption, fluorescence, circular dichroism, optical melting and microcalorimetry have been used.Absorption and fluorescence studies revealed noncooperative 1:1 binding for all the molecules. Coralyne showed highest affinity (10⁶ M⁻¹) and for others it was ~10⁵ M⁻¹. The binding affinity varied as coralyne>sanguinarine>berberine>palmatine. Ethidium showed affinity close to sanguinarine. Comparative fluorescence quenching and polarization anisotropy of the emission spectra gave evidence for a stronger stacking interaction of coralyne and sanguinarine compared to berberine and palmatine. Circular dichroic spectral perturbations were similar in all the cases, but a strong induced circular dichroism for the bound molecules was observed only for coralyne and sanguinarine. The interaction of all the alkaloids was exothermic. Binding of coralyne and sanguinarine was predominantly enthalpy driven while that of berberine and palmatine was entropy driven. Heat capacity values of −169, −198, −105 and −95cal/molK, respectively, for coralyne, sanguinarine, berberine, and palmatine suggested significant differences in the hydrophobic contribution to the binding.This study presents a complete structural and thermodynamic profile of the binding of isoquinoline alkaloids with G-quadruplex.These results suggest strong and specific binding of these molecules to the G-quadruplex and highlight the differences in their structure in the interaction profile.Display Omitted► Comparative studies with DNA intercalator ethidium were also performed. ► Insights into the nature and energetics vis-a-vis structural diversity was obtained. ► This study presents the comparative structural and energetic aspects of binding.

Keywords: Isoquinoline alkaloids; Quadruplex DNA; Human telomeric DNA–alkaloids interaction; Spectroscopy; Calorimetry

Sections

Personal tools

BBA - General Subjects (v.1810, #4)

Sections

Personal tools

Local resources

BBA - General Subjects (v.1810, #4)