BBA - General Subjects (v.1674, #3)

Heterologous expression of a plant arginine decarboxylase gene in Trypanosoma cruzi by Carolina Carrillo; María P. Serra; Claudio A. Pereira; Alejandra Huber; Nélida S. González; Israel D. Algranati (223-230).
Wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity. However, the transformation of these parasites with a recombinant plasmid containing the oat ADC cDNA coding region gave rise to the transient heterologous expression of the enzyme, suggesting the absence of endogenous mechanisms that could inhibit the expression of a hypothetical own ADC gene or the assay used to measure its enzymatic activity. The foreign ADC enzyme expressed in the transgenic T. cruzi was characterized by identification of the products, the stoichiometry of the catalysed reaction, the specific inhibition by α-difluoromethylarginine (DFMA) and the study of its metabolic turnover. The half-life of the heterologous ADC activity in T. cruzi was about 150 min. Bioinformatics studies and polymerase chain reaction (PCR) analyses seem to indicate the absence of ADC-like DNA sequences in the wild-type T. cruzi genome.
Keywords: Arginine decarboxylase; Polyamine biosynthesis; Trypanosoma cruzi epimastigote; Transgenic parasite; Agmatine;

Our findings show a significant increase of free radical production after exposure to 50 Hz electromagnetic fields at a flux density of 1 mT to mouse bone marrow-derived (MBM) promonocytes and macrophages, indicating the cell-activating capacity of extremely low frequency magnetic fields (ELF-MF). We demonstrate that after exposure to ELF-MF mainly superoxide anion radicals were produced, both in MBM macrophages (33%) and also in their precursor cells (24%). To elucidate whether NADPH- or NADH-oxidase functions are target proteins for MF interaction, the flavoprotein inhibitor diphenyleneiodonium chloride (DPI) was used. MF-induced free radical production was not inhibited by DPI, whereas tetradecanoylphorbolacetate (TPA)-induced free radical production was diminished by about 70%. TPA is known to induce a direct activation of NADPH-oxidase through the PKC pathway. Since DPI lacks an inhibitory effect in MF-exposed MBM cells, we suggest that 50 Hz MF stimulates the NADH-oxidase pathway to produce superoxide anion radicals, but not the NADPH pathway. Furthermore, we showed an oscillation (1–10 days) in superoxide anion radical release in mouse macrophages, indicating a cyclic pattern of NADH-oxidase activity.
Keywords: Free radical; ELF-MF; ROS; NAD(P)H-oxidase; NADH-oxidase; Promonocyte macrophage;

Keywords: Endo-β-1,4-mannanase; Cellulose-binding module; Aspergillus fumigatus; Retaining glycoside hydrolase; Transglycosylation;

Do the serum oxidative stress biomarkers provide a reasonable index of the general oxidative stress status? by Sandro Argüelles; Sonia García; Mariam Maldonado; Alberto Machado; Antonio Ayala (251-259).
The oxidant status of an individual is assessed by determining a group of markers in noninvasive samples. One limitation when measuring these biomarkers is that they do not give information about tissue localization of oxidative stress. The present study was undertaken to establish whether the serum oxidative stress biomarkers are indicative of oxidative stress in tissues of an individual. To accomplish this, we determined a few generic markers of oxidation in serum and tissues of six groups of rats treated experimentally, to modulate their oxidative stress status. The correlation between serum and tissue levels was calculated for each marker. Also, for each tissue, the correlation between the values of these oxidative stress biomarkers was analysed. Our results show that only lipid peroxides in serum could be useful to predict the oxidative stress in tissues. No correlation was found between any of the oxidative stress markers in serum.
Keywords: TBARS; Lipid peroxide; Carbonyl group; Antioxidant capacity; Oxidative stress; Serum; Oxidative stress marker;

Iron nitrosyl hemoglobin formation from the reaction of hydroxylamine and hemoglobin under physiological conditions by Virginia L. Lockamy; Howard Shields; Daniel B. Kim-Shapiro; S. Bruce King (260-267).
Sickle cell disease patients receiving hydroxyurea (HU) therapy have shown increases in the production of nitric oxide (NO) metabolites, which include iron nitrosyl hemoglobin (HbNO), nitrite, and nitrate. However, the exact mechanism by which HU forms HbNO in vivo is not understood. Previous studies indicate that the reaction of oxyhemoglobin (oxyHb) or deoxyhemoglobin (deoxyHb) with HU are too slow to account for in vivo HbNO production. In this study, we show that the reaction of methemoglobin (metHb) with HU to form HbNO could potentially be fast enough to account for in vivo HbNO formation but competing reactions of either excess oxyHb or deoxyHb during the reaction reduces the likelihood that HbNO will be produced from the metHb–HU reaction. Using electron paramagnetic resonance (EPR) spectroscopy we have detected measurable amounts of HbNO and metHb during the reactions of oxyHb, deoxyHb, and metHb with excess hydroxylamine (HA). We also demonstrate HbNO and metHb formation from the reactions of excess oxyHb, deoxyHb, or metHb and HA, conditions that are more likely to mimic those in vivo. These results indicate that the reaction of hydroxylamine with hemoglobin produces HbNO and lend chemical support for a potential role for hydroxylamine in the in vivo metabolism of hydroxyurea.
Keywords: Iron nitrosyl hemoglobin; Hydroxylamine; Hemoglobin; Nitric oxide; Hydroxyurea;

Specific xyloglucanases as a new class of polysaccharide-degrading enzymes by Sergey G. Grishutin; Alexander V. Gusakov; Alexander V. Markov; Boris B. Ustinov; Margarita V. Semenova; Arkady P. Sinitsyn (268-281).
Keywords: Endoglucanase; Aspergillus japonicus; Chrysosporium lucknowense; Trichoderma reesei; Xyloglucan; Xyloglucanase;

Specificity of Amaranthus leucocarpus syn. hypocondriacus lectin for O-glycopeptides by Pedro Hernández; Daniel Tetaert; Gérard Vergoten; Henri Debray; Maria del Carmen Jimenez; Georgina Fernández; Concepción Agundis; Pierre Degand; Edgar Zenteno (282-290).
Amaranthus leucocarpus syn. hypochondriacus lectin (ALL) has been shown to be specific for N-acetyl-d-galactosamine (GalNAc). In this work, we determined a value of 1.0×10−2 M for the association constant of ALL for GalNAc, calculated using fluorescence spectroscopy assays. Using neoglycopeptides obtained by in vitro O-glycosylation, we determined the main features of O-glycopeptides recognized by ALL using molecular dynamics simulations, capillary electrophoresis, and ELISA. Neo-glycopeptides were obtained by in vitro O-glycosylation reaction using microsomal preparations of murine thymocytes, human gastric fundus and colonic mucosa. ELISA assays were performed with peroxidase-labeled murine monoclonal IgG2, κ light chain (5D4) antibodies against ALL. Among the in vitro neoglycopeptides, only those of TTSAPTTS containing GalNAc at Thr in #2 and #6 reacted with ALL. Neither the TTSAPTTS glycopeptide, containing a unique GalNAc residue at Thr in #2, nor others (with more than two GalNAc residues) interacted with the lectin. Computational docking assays of the lower energy conformers for interactions between glycopeptides and lectins confirmed that ALL recognized GalNAc residues when they are spaced out in glycan structures, whereas GalNAc residues arranged in clusters prevented interaction with the lectin, indicating that ALL is specific for a special GalNAc-containing motif found in different O-glycoproteins.
Keywords: Amaranthus leucocarpus; Plant lectin; T- and Tn-specific lectin; O-glycan;

The structure–activity relationship of various YO compounds, novel plasmin inhibitors, in the apoptosis induction by Riyo Enomoto; Chiyoko Sugahara; Tomoe Komai; Chie Suzuki; Noriko Kinoshita; Akiko Hosoda; Asa Yoshikawa; Yuko Tsuda; Yoshio Okada; Eibai Lee (291-298).
We have previously reported that YO-2, a selective plasmin inhibitor, induces thymocyte apoptosis. To elucidate the mechanism of YO-2-induced apoptosis, other YO compounds with different plasmin inhibitory action were tested for the pro-apoptotic activity in this study. The treatment of rat thymocytes with the YO compounds which had the hydrophobic but not the hydrophilic moiety at the C-terminal increased DNA fragmentation, the number of condensed nuclei and caspase-3-like activity. All pro-apoptotic YO compounds not only were potent plasmin inhibitors but also had the hydrophobic C-terminal as the common structure. Therefore, the target molecule of the YO compounds may be located not on the cell surface but rather inside the cells.
Keywords: Apoptosis; YO compound; Structure–activity relationship; Plasmin inhibitor; YO-2; Thymocyte;

Molecular association of lectin and β-glucosidase in corn coleoptile by Juan Molina; Abraham Landa; Gonzalo Bautista; Margarito Martínez; Félix Córdoba (299-304).
Corn coleoptile lectin is present with β-glucosidase (EC. in a single tightly bound molecular association complex (88.7 kDa). SDS-PAGE of the molecular complex dissociates into two main components. Of these, at a concentration of 75%, the corn coleoptile β-glucosidase (60 kDa) is identified by enzymatic activity, with two 16-amino acid tryptic peptides displaying close homology with the primary structure of the enzyme. In separate experiments, we isolated homogenous monomeric enzyme of corn coleoptile. This allowed us to conclude that lectin properties like erythrocyte agglutination, found in the (88.7 kDa) molecular complex, is not due to the β-glucosidase bound in it. Another protein (30 kDa) dissociated from the same SDS-PAGE gels rendered several tryptic peptides, including a 20-amino acid sequence V(L)GP(Q)W(A)GGSGGSPVDITAEPQR closely homologous to the putative β-glucosidase aggregating factor (BGAF) precursor described recently. Tryptic peptide SAFTE(A)WN(V)ELK(V) was also present in the BGAF precursor. KFHEQR peptide was not present in BGAF precursor or any other protein sequence examined. Tryptic peptide TYGPFGA showed good homology with the BGAF precursor protein, FEGLYLFHTPLGSGAN peptide displayed identity with the BGAF precursor sequence. Thus, the 30 kDa protein does not appear to be identical to BGAF, but is rather a similar molecule which could be endowed with the lectin properties of the 88.7 kDa molecular complex.
Keywords: Corn coleoptile lectin; β-Glucosidase; β-Glucosidase molecular association; Galactose lectin; Zea mays;

The cytotoxic β-amyloid peptide (Aβ) of Alzheimer's disease (AD) occurs in both plasma and platelets and may modulate platelet function. Its biological activity may relate to its fibril content and factors that promote Aβ fibrillogenesis, e.g., plasma lipoproteins could, therefore, have implications for Aβ action. We undertook a study in which structure–activity relationships were considered with respect to the actions of Aβ1–40 on platelet function. Thus, the influence of soluble Aβ and various fibrillar Aβ preparations (0.1–10 μM) on platelet aggregation and endogenous 5-hydroxytryptamine (5-HT) efflux was investigated. Soluble Aβ1–40 only enhanced platelet aggregation (+30%, P<0.05) and 5-HT release (+28%) stimulated by ADP (1 μM) at the highest concentration tested (10 μM). By contrast, fibrillar Aβ1–40 at 1, 5 and 10 μM potentiated aggregation by 17.4%, 68.8% (P<0.05) and 99.5% (P<0.0001), respectively, and 5-HT efflux by 17.4%, 65% and 208% (P<0.001). Aβ1–40 fibrils generated in the presence of native and oxidised very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) yielded platelet responses that did not differ from those seen with the lipoproteins alone. These responses were markedly lower than those obtained with homogeneous Aβ fibrils. Our data indicate that homogeneous Aβ1–40 fibrils are more potent than soluble Aβ1–40 in promoting platelet reactivity and that interactions with plasma lipoproteins result in the formation of Aβ fibrils that are ineffective. We suggest that lipoproteins may interfere with the recognition of Aβ by appropriate platelet receptors and/or cause Aβ to assume an “overaggregated” biologically inert state.
Keywords: β-Amyloid; Plasma lipoprotein; Fibrillogenesis; Platelet aggregation;

Inhibitory effects of trientine, a copper-chelating agent, on induction of DNA strand breaks in hepatic cells of Long–Evans Cinnamon rats by Masanobu Hayashi; Kazuhiro Miyane; Takeshi Hirooka; Daiji Endoh; Hidetoshi Higuchi; Hajime Nagahata; Kenji Nakayama; Yashuhiro Kon; Toyo Okui (312-318).
Effects of treatment with trientine, a specific copper-chelating agent, on accumulation of copper and induction of DNA strand breaks were investigated in Long–Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. Copper accumulated in the livers of LEC rats in an age-dependent manner from 4 to 13 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, hepatic copper contents did not increase and were maintained at the same levels as those in 10-week-old LEC rats. When the amounts of DNA single-strand breaks (SSBs) were estimated by a comet assay, SSBs of DNA were induced in a substantial population of LEC rat hepatic cells around 8 weeks of age and the amounts of SSBs increased in an age-dependent manner from 8 to 15 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, the observed number of cells with DNA damage decreased dramatically, suggesting that induction of SSBs of DNA was inhibited and/or SSBs were repaired during the period of treatment with trientine. The results show that treatment of LEC rats with trientine decreases the number of DNA strand breaks observed, although copper contents remain high in the liver.
Keywords: Copper; DNA strand break; Hepatic cell; LEC rat; Trientine;

Cytochrome b 5 (cyt b 5) is an amphipathic membrane-bound heme protein found in the endoplasmic reticulum of eukaryotes. It consists of three domains, an N-terminal cytosolic, hydrophilic domain containing the heme, a short flexible linker and an α-helical membrane-spanning domain. This study investigated whether there are specific side chain helix–helix packing interactions between the COOH-terminal membrane anchor of cyt b 5 and cytochrome P450 (cyt P450) 2B4 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b 5. Insertion of alanine into an α-helix causes all amino acids at its carboxyl terminus to be rotated by 100°. The ability of the alanine insertion mutants of cyt b 5 to bind to cyt P450 2B4 was similar to that of the wild-type protein as was the ability of the mutant cyts b 5 to stimulate the metabolism of the anesthetic, methoxyflurane. These results demonstrate that the C-terminal hydrophobic α-helix of cyt b 5 does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions.
Keywords: Cytochrome b 5; Cytochrome P450 2B4; Mutagenesis; Membrane protein; Protein–protein interaction;

Author Index (327-329).

Cumulative Contents (330-332).