BBA - General Subjects (v.1528, #1)

Nitric oxide (NO) generated by the reduction of sodium nitrite with sodium dithionite caused damage to plasmid Bluescript DNA leading to strand breaks and base modifications. The NO-plasmid DNA was highly immunogenic in rabbits. The antibody activity was inhibited to the extent of 86% with the immunogen as inhibitor, indicating the induction of immunogen specific antibodies. However, delineating the antigenic specificity of anti-NO-plasmid DNA antibodies by competition ELISA, multiple cross-reactivity was observed. The antibodies recognised B-, A- and allied conformations. The visual detection of immune complex formation with native and NO-plasmid DNA reiterated preferential binding with modified plasmid DNA. DNA modified by nitric oxide presents unique epitopes which may be one of the factors in antigen-driven autoimmune response in systemic lupus erythematosus.
Keywords: Nitric oxide-plasmid DNA; Nitric oxide; Systemic lupus erythematosus; Anti-DNA antibody; Autoantibody;

A novel disaccharide substrate having 1,2-oxazoline moiety for detection of transglycosylating activity of endoglycosidases by Masaya Fujita; Shin-ichiro Shoda; Katsuji Haneda; Toshiyuki Inazu; Kaoru Takegawa; Kenji Yamamoto (9-14).
A disaccharide substrate of Manβ1-4GlcNAc-oxazoline 2 was designed and synthesized as a novel probe for detection of the transglycosylating activity of endoglycosidases. A regio- and stereoselective transglycosylation reaction of 2 to GlcNAcβ1-O-pNP or Dns-Asn(GlcNAc)-OH catalyzed by endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) and endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has been demonstrated for the first time, resulting in the core trisaccharide derivative Manβ1-4GlcNAcβ1-4GlcNAcβ1-O-pNP 8 (or -(Dns)Asn-OH). Interestingly, the transglycosylation proceeds irreversibly; the resulting trisaccharide 8 was not hydrolyzed by Endo-M and Endo-A. Based on these results, a new mechanism including an oxazolinium ion intermediate has been proposed for the endoglycosidase-catalyzed hydrolysis or transglycosylation.
Keywords: Sugar oxazoline; Transition state analogue substrate; Endo-β-N-acetylglucosaminidase; Transglycosylation; Regioselective glycosylation; Stereoselective glycosylation; Oxazolinium ion intermediate;

In aqueous solutions N-methyl-N-d-fructosyl amphotericin B methyl ester (MFAME), a novel amphotericin B derivative with low animal toxicity, similar to its parent antibiotic, exists in three forms: monomeric, soluble and insoluble aggregates in equilibrium . The aim of our work was to examine the influence of medium composition on the MFAME self-association and the relationship between MFAME self-association and its toxicity towards red blood cells. The toxicity of MFAME in aggregated state towards red blood cells was tested by measuring the induction of potassium leakage and extent of haemolysis. The proportions of antibiotic species present in various aqueous media were determined by analysis of the UV-Vis spectra as a function of the antibiotic concentration. Numeric decomposition of the spectra allowed identification of four spectral species present in MFAME solutions: monomeric and three aggregated forms. Our results indicate that these aggregates, named type I, type II and type III, are different in terms of spectral properties, as well as effectiveness towards red blood cells. Soluble aggregate types I and III are the active forms of MFAME towards erythrocytes. The medium composition seems to be the main factor determining which type of antibiotic aggregate prevails in solution.
Keywords: Amphotericin B; Amphotericin B derivative; Rational drug design; Self association; Selective toxicity;

Superoxide is responsible for apoptosis in rat vascular smooth muscle cells induced by α-tocopheryl hemisuccinate by Kentaro Kogure; Motoki Morita; Sawa Nakashima; Susumu Hama; Akira Tokumura; Kenji Fukuzawa (25-30).
We investigated the mechanism of cell toxicity of α-tocopheryl hemisuccinate (TS). TS concentration- and time-dependently induced the lactate dehydrogenase release and DNA fragmentation of rat vascular smooth muscle cells (VSMC). Exogenous addition of superoxide dismutase, but not catalase, significantly inhibited the cell toxicity of TS. The NADPH-dependent oxidase activity of VSMC was stimulated by TS treatment. The cell toxicity of TS was inhibited by NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride. Consequently, TS-induced apoptosis of VSMC was suggested to be caused by exogenous O2 generated via the oxidase system activated with TS.
Keywords: α-Tocopheryl hemisuccinate; Apoptosis; Superoxide; Vascular smooth muscle cell;

Quantitative structure–activity relationships in enzymatic single-electron reduction of nitroaromatic explosives: implications for their cytotoxicity by Narimantas Č≐nas; Aušra Nemeikait≐-Č≐nien≐; Egl≐ Sergedien≐; Henrikas Nivinskas; Žilvinas Anusevičius; Jonas Šarlauskas (31-38).
The mechanisms of cytotoxicity of polynitroaromatic explosives, an important group of environmental pollutants, remain insufficiently studied so far. We have found that the rate constants of single-electron enzymatic reduction, and the enthalpies of single-electron reduction of nitroaromatic compounds (ΔHf(ArNO2 −⋅)), obtained by quantum mechanical calculation, may serve as useful tools for the analysis of cytotoxicity of nitroaromatic explosives with respect to the possible involvement of oxidative stress. The single-electron reduction rate constants of a number of explosives including 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), and model nitroaromatic compounds by ferredoxin:NADP+ reductase (FNR, EC 1.18.1.2) and NADPH:cytochrome P-450 reductase (P-450R, EC 1.6.2.4) increased with a decrease in ΔHf(ArNO2 −⋅). This indicates that the reduction rates are determined by the electron transfer energetics, but not by the particular structure of the explosives. The cytotoxicity of explosives to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) increased with a corresponding increase in their reduction rate constant by P-450R and FNR, or with a decrease in their ΔHf(ArNO2 −⋅). This points to an importance of oxidative stress in the toxicity of explosives in this cell line, which was further evidenced by the protective effects of desferrioxamine and the antioxidant N,N′-diphenyl-p-phenylene diamine, and an increase in lipid peroxidation. DT-diaphorase (EC 1.6.99.2) exerted a minor and equivocal role in the cytotoxicity of explosives to FLK cells.
Keywords: TNT; Tetryl; Pentryl; Redox cycling; Cytotoxicity; Ferredoxin:NADP+ reductase; NADPH:cytochrome P-450 reductase; DT-diaphorase;

Effect of tunicamycin on N-acetyl-β-d-glucosaminidase produced by Trichoderma harzianum by C.J. Ulhoa; D. Sankievicz; P.S. Limeira; J.F. Peberdy (39-42).
The effect of tunicamycin, an inhibitor of protein N-glycosylation, was studied in non-growing mycelium of Trichoderma harzianum induced to secrete N-acetyl-β-d-glucosaminidase by the addition of N-acetylglucosamine. Tunicamycin (30 μg ml−1) had no significant effect on growth of the fungus, or on the total protein secreted or specific activity of N-acetyl-β-d-glucosaminidase. However, in the presence of the inhibitor an underglycosylated form of the enzyme was produced. The apparent molecular masses for this and the native enzyme were 110 and 124 kDa, respectively. Both forms of the enzyme showed the same optimum pH and temperature, but the underglycosylated form was more sensitive to inactivation by both high temperature (60°C) and the proteolytic enzyme trypsin.
Keywords: N-Acetyl-β-d-glucosaminidase; Glycosylation; Tunicamycin; Trichoderma harzianum;

Little is known about the acquisition of cobalamin by the mammary gland and its secretion into milk. Human milk and plasma contain at least two types of cobalamin binding proteins: transcobalamin II (TC) and haptocorrin (HC). In plasma, TC is responsible for the transport of cobalamin to tissues and cells; however, cobalamin in milk is present exclusively bound to HC. We show that human mammary epithelial cells (HMEC) exhibit high affinity for TC; Scatchard analysis revealed a single class of binding sites for the TC–[57Co]cyanocobalamin complex with a dissociation constant (K d) of 4.9×10−11 M. Uptake of the TC–[57Co]cyanocobalamin complex at 37°C was saturable by 24 h. Binding of free [57Co]cyanocobalamin to HMEC was not saturable and very limited binding of the HC–[57Co]cyanocobalamin complex was observed. Expression of the haptocorrin gene by HMEC was confirmed by Northern blot and PCR analysis. Thus, a specific cell surface receptor for the TC–cobalamin complex exists in the mammary gland and once cobalamin is internalized, it may be transferred to HC and subsequently secreted into milk as a HC–cobalamin complex.
Keywords: Transcobalamin; Transcobalamin II receptor; Haptocorrin; Vitamin B12; Cobalamin; Mammary gland;

Quercetin may contribute to the protection afforded by fruit- and vegetable-rich diets against diseases for which excess production of reactive oxygen species (ROS) has been implicated as a causal or contributory factor. We examine the effect of short term (90 min) quercetin (1–100 μM) exposure on the progress of menadione induced oxidative stress within HL-60 cells. 2′,7′-dichlorofluorescein and rhodamine-123 fluorescence, resulting from oxidation of the ROS-sensitive dyes dichlorodihydrofluorescein and dihydrorhodamine-123 respectively, were utilised as indicators of general ROS levels. Ethidium fluorescence, resulting from oxidation of dihydroethidium, was used as a potentially more specific indicator of O2 . Exposure to quercetin alone induced a decrease in DCF and rhodamine fluorescence. Conversely, ethidium fluorescence was enhanced by treatment with ≥40 μM quercetin. Incubation with 1–100 μM quercetin reduced the extent of menadione-induced increase in DCF and rhodamine fluorescence but the menadione-induced increase in ethidium fluorescence was further elevated for cells treated with ≥25 μM quercetin. Exposure to ≥10 μM quercetin abrogated menadione-induced DNA single-strand breaks but, paradoxically, quercetin exacerbated membrane damage and failed to enhance the viability of menadione-challenged cells. In conclusion, quercetin exerts only site-specific protection against oxidative stress.
Keywords: Flavonoid; HL-60; Menadione; Oxidative stress; Quercetin; Cellular integrity;