BBA - General Subjects (v.1810, #8)

Broadband dielectric spectroscopy on human blood by M. Wolf; R. Gulich; P. Lunkenheimer; A. Loidl (727-740).
Dielectric spectra of human blood reveal a rich variety of dynamic processes. Achieving a better characterization and understanding of these processes not only is of academic interest but also of high relevance for medical applications as, e.g., the determination of absorption rates of electromagnetic radiation by the human body.The dielectric properties of human blood are studied using broadband dielectric spectroscopy, systematically investigating the dependence on temperature and hematocrit value. By covering a frequency range from 1 Hz to 40 GHz, information on all the typical dispersion regions of biological matter is obtained.We find no evidence for a low-frequency relaxation (“α-relaxation”) caused, e.g., by counterion diffusion effects as reported for some types of biological matter. The analysis of a strong Maxwell–Wagner relaxation arising from the polarization of the cell membranes in the 1–100 MHz region (“β-relaxation”) allows for the test of model predictions and the determination of various intrinsic cell properties. In the microwave region beyond 1 GHz, the reorientational motion of water molecules in the blood plasma leads to another relaxation feature (“γ-relaxation”). Between β- and γ-relaxations, significant dispersion is observed, which, however, can be explained by a superposition of these relaxation processes and is not due to an additional “δ-relaxation” often found in biological matter.Our measurements provide dielectric data on human blood of so far unsurpassed precision for a broad parameter range. All data are provided in electronic form to serve as basis for the calculation of the absorption rate of electromagnetic radiation and other medical purposes. Moreover, by investigating an exceptionally broad frequency range, valuable new information on the dynamic processes in blood is obtained.Display Omitted► Dielectric spectra of human blood in dependence of temperature and hematocrit value. ► Broad frequency range (1 Hz–40 GHz) enabling detection of several dynamic processes. ► Detection of beta-, gamma-, and delta-dispersion. ► Calculation of dielectric properties of blood cells. ► Basis for calculation of absorption rates of electromagnetic radiation is provided.
Keywords: Dielectric spectroscopy; Blood; Relaxation; Specific absorption rate; Dielectric loss; Dielectric constant;

New studies provide evidence that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is not simply a classical glycolytic protein of little interest. Instead, it is a multifunctional protein with significant activity in a number of fundamental cell pathways. GAPDH is a highly conserved gene and protein, with a single mRNA transcribed from a unique gene. Control mechanisms must exist which regulate its functional diversity.This review focuses on new, timely studies defining not only its diverse activities but also those which define the regulatory mechanisms through which those functions may be controlled. The reader is referred to the author's prior review for the consideration of past reports which first indicated GAPDH multiple activities (Sirover, Biochim. Biophys. Acta 1432 (1999) 159–184.)These investigations demonstrate fundamental roles of GAPDH in vivo, dynamic changes in its subcellular localization, and the importance of posttranslational modifications as well as protein:protein interactions as regulatory control mechanisms.GAPDH is the prototype “moonlighting” protein which exhibits activities distinct from their classically identified functions. Their participation in diverse cell pathways is essential. Regulatory mechanisms exist which control those diverse activities as well as changes in their subcellular localization as a consequence of those new functions.► GAPDH is a multidimensional protein with functions distinct from glycolysis. ► Its independent activities depend on its dynamic subcellular translocation. ► Its functional diversity is regulated posttranslationally. ► Multiple mechanisms may be required for its nuclear translocation.
Keywords: Glyceraldehyde-3-phosphate dehydrogenase; Multifunctional protein; Subcellular translocation;

A HA2-Fusion tag limits the endosomal release of its protein cargo despite causing endosomal lysis by Ya-Jung Lee; Gregory Johnson; Grantham C. Peltier; Jean-Philippe Pellois (752-758).
Protein transduction domains (PTDs) can be fused to a protein to render it cell-permeable. The delivery efficiencies of PTDs are, however, often poor because PTD-protein conjugates cannot escape from endosomes. A potential solution to this problem consists in adding HA2 analogs to the PTD-protein construct as these peptides can cause endosomal lysis upon acidification of the endosomal lumen. To date, however, the utility of HA2-based PTDs has not been clearly established.We investigate the biophysical and cellular properties of the glutamate-rich HA2 analog E5 fused to the model protein TAT-mCherry.E5-TAT-mCherry causes the release of fluorescent dextrans trapped with the protein inside endosomes. Yet, E5-TAT-mCherry itself is not released in the cytosol of cells, indicating that the protein remained trapped inside endosomes even after endosomal lysis takes place. Cytosolic delivery of the protein could be achieved, however, by insertion of a disulfide bond between E5 and its cargo.These results show that E5 causes the retention of its fused protein inside endosomes even after lysis takes place.These data establish that HA2 analogs might not be useful PTDs unless cleavable linkers are engineered between PTD and protein cargo.► We investigate the delivery of mCherry fused to the endosomolytic peptide E5. ► E5 causes the lysis of endosomes upon acidification of the endosomal lumen. ► mCherry fused to E5 cannot escape from lysed endosomes. ► mCherry attached to E5 through a disulfide bond can escape from lysed endosomes. ► A cleavable linker between E5 and its cargo is required to increase endosomal escape.
Keywords: Protein delivery; HA2 peptide; Endosomal escape; Protein transduction domain; Lysis;

Ganglioside GD1a negatively regulates hepatocyte growth factor expression through caveolin-1 at the transcriptional level in murine osteosarcoma cells by Lan Zhang; Yinan Wang; Li Wang; Ting Cao; Sumiko Hyuga; Toshinori Sato; YingLiang Wu; Sadako Yamagata; Tatsuya Yamagata (759-768).
Hepatocyte growth factor (HGF) is a mesenchyme-derived, multifunctional protein that is implicated in tumor growth and invasive behavior. Some tumor cells express both HGF and its receptor MET, forming an autocrine loop that permanently activates it. Ganglioside GD1a suppresses metastatic capacity in murine FBJ osteosarcoma cells and MET phosphorylation activated by HGF binding, but the signaling pathway controlling HGF production has not been fully explored.Expression of HGF, caveolins, or MET of the cells that had been transfected with siRNA or cDNA directed to GM2/GD2 synthase, caveolin-1 or HGF was determined by semi-quantitative RT-PCR and Western blots.HGF expression in highly metastatic, GD1a-deficient FBJ-LL cells was higher than that in the poorly metastatic, GD1a-rich FBJ-S1 cells. Transfection with GM2/GD2 synthase cDNA increased GD1a levels in FBJ-LL cells and suppressed HGF expression. Treatment with siRNAs directed toward GM2/GD2 synthase in FBJ-S1 cells reduced gangliosides and augmented HGF expression. GD1a was found to be the only ganglioside species suppressing HGF expression upon addition to FBJ-LL cells. HGF expression was decreased by GD1a addition to FBJ-LL cells after 48 h, enough to induce caveolin-1 expression. Silencing caveolin-1 up-regulated HGF, and the re-introduction of caveolin-1 cDNA decreased HGF expression. Caveolin-1 suppressed MET phosphorylation. We also found GD1a regulation of HGF in Lewis lung carcinoma cells.HGF expression was negatively regulated by GD1a through caveolin-1 at the transcriptional level via the suppression of MET phosphorylation.This is the first report that ganglioside GD1a negatively regulates HGF expression through caveolin-1.► Ganglioside GD1a was shown to suppress HGF expression at the transcriptional level. ► GD1a increases caveolin-1 expression that also negatively regulates HGF expression. ► Caveolin-1 suppressed MET phosphorylation. ► GD1a regulation of HGF was also found in Lewis lung carcinoma cells.
Keywords: Ganglioside GD1a; Hepatocyte growth factor; MET; Metastasis; Caveolin-1; FBJ cells; Osteosarcoma;

Acridine and quindoline oligomers linked through a 4-aminoproline backbone prefer G-quadruplex structures by Rubén Ferreira; Roberto Artali; Josep Farrera-Sinfreu; Fernando Albericio; Miriam Royo; Ramon Eritja; Stefania Mazzini (769-776).
DNA-intercalating drugs are planar molecules with several fused aromatic rings that form stacks between DNA base pairs, reducing the opening and unwinding of the double helix. Recently, interest on intercalating agents has moved in the search for new ligands to G-quadruplex structures.The DNA binding properties of 4-aminoproline oligomers functionalized with one, two or three units of acridine and/or quindoline have been analyzed by competitive dialysis. A NMR/molecular dynamics study was performed on G-quadruplex telomeric sequence and the 4-aminoproline dimer carrying two quindolines. A model of the complex with the telomeric DNA quadruplex is described.A selectivity of quindoline 4-aminoproline oligomers for G-quadruplex and triplex structures was observed, especially for those quadruplex sequences found in telomeres and in the promoter regions of c-myc and bcl-2 oncogenes. In this model the quindoline dimer is stabilized by π–π stacking interactions between the aromatic rings of the ligand and the nucleobases of the telomeric sequence that are located above and below the molecule.The results of this work can be used for the design of new molecules with high affinity to telomeres which may have anticancer properties.Display Omitted ► Selectivity of quindoline 4-aminoproline for G-quadruplex and triplex structures. ► NMR/molecular dynamics study on G-quadruplex telomeric and the 4-aminoproline dimer. ► In G-quadruplex model the quindoline dimer is stabilized by π–π stacking.
Keywords: Oligonucleotides; Acridine; Quindoline; G-quadruplex; NMR; DNA-binding drugs;

Amphotericin B induces trehalose synthesis and simultaneously activates an antioxidant enzymatic response in Candida albicans by Pilar González-Párraga; Ruth Sánchez-Fresneda; Óscar Zaragoza; Juan-Carlos Argüelles (777-783).
Enzymes involved in trehalose metabolism have been proposed as potential targets for new antifungals. To analyse this proposal, the susceptibility to Amphotericin B (AmB) of the C. albicans trehalose-deficient mutant tps1Δ/tps1Δ, was examined.Determination of endogenous trehalose and antioxidant enzymatic activities as well as RT-PCR analysis in cells subjected to AmB treatments was performed.Exponential tps1Δ null cultures showed high degree of cell killing upon exposure to increasing AmB doses respect to CAI.4 parental strain. Reintroduction of the TPS1 gene restored the percentage of cell viability. AmB induced significant synthesis of endogenous trehalose in parental cells, due to the transitory accumulation of TPS1 mRNA or to the moderate activation of trehalose synthase (Tps1p) with the simultaneous deactivation of neutral trehalase (Ntc1p). Since tps1Δ/tps1Δ mutant cells are highly susceptible to acute oxidative stress, the putative antioxidant response to AmB was also measured. A conspicuous activation of catalase and glutathione reductase (GR), but not of superoxide dismutase (SOD), was observed when the two cell types were exposed to high concentrations of AmB (5 μg/ml). However, no significant differences were detected between parental and tps1Δ null strains as regards the level of activities.The protective intracellular accumulation of trehalose together with the induction of antioxidant enzymatic defences are worthy mechanisms involved in the resistance of C. albicans to the fungicidal action of AmB.The potential usefulness of trehalose synthesis proteins as an interesting antifungal target is reinforced. More importantly, AmB elicits a complex defensive response in C. albicans.► Candida albicans evolves a complex defensive response against Amphotericin B. ► Amphotericin B triggers trehalose storage which depends on functional TPS1 gene. ► A conspicuous activation of catalase and gluthatione reductase were also recorded. ► Trehalose and antioxidant induction participate in the resistance to Amphotericin B.
Keywords: Amphotericin B; Antioxidant enzymes; Trehalose; Candida albicans;

Metabolite modulation of HeLa cell response to ENOX2 inhibitors EGCG and phenoxodiol by Lian-Ying Wu; Thomas De Luca; Takahiro Watanabe; Dorothy M. Morré; D. James Morré (784-789).
Constituents and inhibitors of intermediary metabolism resulting in alterations in levels of cytosolic NADH, stimulation of sphingomyelinase and inhibition of sphingosine kinase were evaluated for effects on growth inhibition and induction of apoptosis by the ENOX2 inhibitors EGCG, the principal catechin of green tea, and phenoxodiol, a naturally occurring isoflavone.Responses were evaluated from dose–response curves of the metabolites and metabolic inhibitors in which growth of HeLa cells, apoptosis based on DAPI fluorescence and cytosolic NADH levels were correlated with sphingomyelinase and spingosine kinase activities and levels of ceramide and sphingosine1-phosphate.Growth inhibition correlated with the modulation of localized cytosolic NADH levels by metabolites and metabolic inhibitors, the response of sphingomyelinase and sphingosine kinase located near the inner surface of the plasma membrane, and apoptosis.Based on findings with metabolites, we conclude that apoptosis in cancer cell lines caused by ENOX2 inhibitors such as EGCG and phenoxodiol is a direct response to elevated levels of cytosolic NADH that result from ENOX2 inhibition.The findings help to explain why increased NADH levels resulting from ENOX2 inhibition result in decreased prosurvival sphingosine-1-phosphate and increased proapoptotic ceramide, both of which may be important to initiation of the ENOX2 inhibitor-induced apoptotic cascade.► ENOX2 is a cancer-specific terminal oxidase of mammalian plasma membrane redox. ► When blocked by inhibitors EGCG or phenoxodiol, cytosolic NADH levels increase. ► Increased NADH inhibits prosurvival sphingosine kinase. ► Increased cytosolic NADH levels activate proapoptotic sphingomyelinase. ► Metabolites induce apoptosis in direct proportion to NADH levels.
Keywords: ECTO-NOX2 (tNOX); Metabolite; Phenoxodiol; (−)-Epigallocatechin-3-gallate (EGCG); Sphingolipid; HeLa cell;

ER-stress-inducible Herp, facilitates the degradation of immature nicastrin by Toshihiro Marutani; Tomoji Maeda; Chiaki Tanabe; Kun Zou; Wataru Araki; Koichi Kokame; Makoto Michikawa; Hiroto Komano (790-798).
Herp is an endoplasmic reticulum (ER)-stress-inducible membrane protein harboring an ubiquitin-like domain (ULD). However, its biological functions are not fully understood. Here, we examined the role of Herp in the degradation of γ-secretase components.Effects of ULD-lacking Herp (ΔUb-Herp) expression on the degradation of γ-secretase components were analyzed.The cellular expression of ΔUb-Herp was found to inhibit the degradation of overexpressed immature nicastrin and full-length presenilin. The mechanisms underlying Herp-mediated nicastrin degradation was further analyzed. We found that immature nicastrin accumulates in the ER of ΔUb-Herp overexpressing cells or Herp-deficient cells more than that in the ER of wild-type cells. Further, ΔUb-Herp expression inhibited nicastrin ubiquitination, suggesting that the ULD of Herp is likely involved in nicastrin ubiquitination. Co-immunoprecipitation study showed that Herp as well as ΔUb-Herp potentially interacts with nicastrin, mediating nicastrin interaction with p97, which functions in retranslocation of misfolded proteins from the ER to the cytosol.Thus, Herp is likely involved in degradation of immature nicastrin by facilitating p97-dependent nicastrin retranslocation and ubiquitination. General significance: We suggest that Herp could play a role in the elimination of the excess unassembled components of a multimeric complex.► The deletion of the ubiquitin-like domain of Herp impairs the degradation of nicastrin. ► Herp facilitates the ubiquitination of nicastrin. ► Herp plays a role for the elimination of the excess unassembled components of a multimeric complex.
Keywords: γ-secretase; Presenilin; Nicastrin; E3 ubiquitin ligase;

Structure–activity relationships of various amino-hydroxy-benzenesulfonic acids and sulfonamides as tyrosinase substrates by Antonio Rescigno; Frédéric Bruyneel; Alessandra Padiglia; Francesca Sollai; Andrea Salis; Jaqueline Marchand-Brynaert; Enrico Sanjust (799-807).
o-Aminophenols have been long recognised as tyrosinase substrates. However their exact mode of interaction with the enzyme's active site is unclear. Properly vic-substituted o-aminophenols could help gain some insight into tyrosinase catalytic mechanism.Eight vic-substituted o-aminophenols belonging to two isomeric series were systematically evaluated as tyrosinase substrates and/or activators and/or inhibitors, by means of spectrophotometric techniques and HPLC-MS analysis. Some relevant kinetic parameters have also been obtained.Four o-aminophenolic compounds derived from 3-hydroxyorthanilic acid (2-amino-3-hydroxybenzenesulfonic acid) and their four counterparts derived from the isomeric 2-hydroxymetanilic acid (3-amino-2-hydroxybenzenesulfonic acid) were synthesised and tested as putative substrates for mushroom tyrosinase. While the hydroxyorthanilic derivatives were quite inactive as both substrates and inhibitors, the hydroxymetanilic compounds on the contrary all acted as substrates for the enzyme, which oxidised them to the corresponding phenoxazinone derivatives.Based on the available structures of the active sites of tyrosinases, the different affinities of the four metanilic derivatives for the enzyme, and their oxidation rates, we propose a new hypothesis regarding the interaction between o-aminophenols and the active site of tyrosinase that is in agreement with the obtained experimental results.Display Omitted► Two series of vic-substituted o-aminophenols were tested as tyrosinase substrates. ► Only hydroxymetanilic compounds were oxidised by the enzyme. ► A rationale for enzyme/substrate interaction in catalytic cycle was proposed.
Keywords: Tyrosinase activity; Active site; Benzenesulfonic acid; Benzenesulfonamide; Phenoxazinone;