BBA - General Subjects (v.1622, #2)

Photodynamic effects of two hydroxyanthraquinones by M. Rajendran; S. Ramasamy; C. Rajamanickam; R. Gandhidasan; R. Murugesan (65-72).
The aim of this work was to investigate the photodynamic action of electron-rich anthraquinones, viz., cynodontin (CYN) and cynodontin-5,8-dimethylether (CYNM). Both optical and EPR methods are used to detect the generation of singlet oxygen. Based on RNO bleaching, relative to rose bengal (RB), singlet oxygen generating efficiencies of CYN and CYNM are derived to be 0.055 and 0.254, respectively. The formation of superoxide anion via electron transfer to O2 was monitored by optical spectroscopy, using SOD-inhibitable cytochrome c reduction assay. The production of O2 −• is enhanced in the presence of electron donors such as EDTA and NADH. Photolysis of CYN and CYNM in DMSO, in the presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), generates a multi-line EPR spectrum, characteristic of spin adduct mixture of O2 −• and OH. Both optical and ESR measurements indicate that O2 −• (Type I) and 1O2 (Type II) paths are involved in CYN and CYNM photodynamic action.
Keywords: Anthraquinone; 5,5-dimethyl-1-pyrroline-N-oxide (DMPO); Singlet oxygen; Superoxide anion; Spin trapping;

Transcriptional regulation of the c-myc gene is essential for normal cellular proliferation; differentiation and overexpression of c-myc is associated with several human cancers. C-myc gene, particularly exon 1, which contains the conserved P1 and P2 promoter regions, has been a potential target for the intercalating drugs in chemotherapy. We have chosen a 21-mer GC-rich oligonucleotide sequence starting from 2281 to 2302 of human c-myc gene located 26 base pair upstream of P1 promoter and partially overlapping with the TATA box of P1. In this paper, we have studied the interaction of a tetrapeptide, KWGK-otBut, with duplex of the above 21-mer sequence under low-salt conditions using UV–Vis absorption, UV melting, fluorescence and circular dichroic (CD) spectroscopy. From the fluorescence quenching data, we determined the two binding constants, K 1 (involving only electrostatic interactions) and K 2 (involving intercalation), for the formation of (PN)1 and (PN)2 of the two-step mechanism previously established by us. Significant changes were observed in the UV difference absorption spectra and CD spectra of both KWGK and 21-mer duplex upon complex formation even at a very low peptide to nucleotide (P/N) ratios. These spectral changes accompanied by a high value of K 2 (=5.13) suggest a strong binding of KWGK involving intercalation of the tryptophan in 21-mer duplex. Based on the above data along with changes observed in ΔH, ΔS and ΔG and increase in melting temperature (by about 8 °C) of the 21-mer duplex in presence of KWGK, we propose a model for intercalation of tryptophan of in GC-rich region of c-myc gene. Present observations may be explored in understanding the role of intercalation in protein–nucleic acid interactions in c-myc expression and these results could also help in designing oligopeptides or other low molecular weight ligands to modulate gene expression.
Keywords: Tryptophan intercalation; c-myc; Binding studies; Peptide; Oligonucleotide;

Streptomyces lividans acetylxylan esterase removes the 2- or 3-O-acetyl groups from methyl 2,4-di-O-acetyl- and 3,4-di-O-acetyl β-d-xylopyranoside. When the free hydroxyl group was replaced with a hydrogen or fluorine, the rate of deacetylation was markedly reduced, but regioselectivity was not affected. The regioselectivity of deacetylation was found to be independent of the prevailing conformation of the substrates in solution as determined by 1H-NMR spectroscopy. These observations confirm the importance of the vicinal hydroxyl group and are consistent with our earlier hypothesis that the deacetylation of positions 2 and 3 may involve a common ortho-ester intermediate. Another possible role of the free vicinal hydroxyl group could be the activation of the acyl leaving group in the deacetylation mechanism. Involvement of the free hydroxyl group in the enzyme–substrate binding is not supported by the results of inhibition experiments in which methyl 2,4-di-O-acetyl β-d-xylopyranoside was used as substrate and its analogues or methyl β-d-xylopyranoside as inhibitors. The enzyme requires for its efficient action the trans arrangement of the free and acetylated hydroxyl groups at positions 2 and 3.
Keywords: Acetylxylan esterase; Streptomyces lividans; Deacetylation of carbohydrate;

Protein kinase C inhibits the transplasma membrane influx of Ca2+ triggered by 4-aminopyridine in Jurkat T lymphocytes by Élie Barbar; Marek Rola-Pleszczynski; Marcel D. Payet; Gilles Dupuis (89-98).
4-Aminopyridine (4AP) is a general blocker of voltage-dependent K+ channels. This pyridine derivative has also been shown to inhibit T cell proliferation, to modulate immune responses and to alleviate some of the symptoms associated with neurological disorders such as multiple sclerosis, myasthenia gravis and Alzheimer's disease. 4AP triggers a Ca2+ response in lymphocytes, astrocytes, neurons and muscle cells but little is known about the regulation of the 4AP response in these cells. We report that 4AP induced a non-capacitative transplasma membrane influx of Ca2+ in Jurkat T lymphocytes. The influx of Ca2+ was not affected by activation or inhibition of protein kinase A (PKA). In contrast, activation of protein kinase C (PKC) by phorbol myristyl acetate (PMA), mezerein or 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited the influx of Ca2+ triggered by 4AP. The inhibitory effect of PKC could be prevented by prior exposure of the cells to the PKC inhibitor GF 109203X. Under these conditions, mezerein and OAG no longer inhibited the 4AP-dependent Ca2+ response. Inhibition of serine and threonine protein phosphatases PP1 and PP2A by treating the cells with calyculin A (CalA) reduced the Ca2+ response to 4AP. Okadaic acid (OA) had no effect, suggesting an involvement of PP1. A combination of CalA and OAG (or PMA) abolished the influx of Ca2+ induced by 4AP, adding further evidence to the importance of protein phosphorylation in the modulation of the 4AP response. Our data suggest that the transplasma membrane influx of Ca2+ triggered by 4AP in Jurkat T cells can be modulated by the opposite actions of PKC and protein serine and threonine phosphatase(s).
Keywords: 4-Aminopyridine; Channel blocker; Calcium; Jurkat cell; Protein kinase C;

Structure determination, apoptosis induction, and telomerase inhibition of CFP-2, a novel lichenin from Cladonia furcata by Xin Lin; Yu-Jun Cai; Zhi-Xiao Li; Qian Chen; Zhong-Li Liu; Rui Wang (99-108).
A great deal of experimental evidence has accumulated in the past several decades, suggesting that polysaccharides have wide bioactivities. Cladonia furcata polysaccharide, CFP-2, a water-soluble lichenin with a mean M r 7.6×104, was first obtained by 0.25 M NaOH solution extraction, ethanol precipitation, DEAE-cellulose, and Sephadex G-200 column chromatography. Gas chromatography of acid hydrolyzate of CFP-2 suggested that it was composed of d-glucose, d-galactose, and d-mannose in the molar ratios of 8:1:1. Periodate oxidation, Smith degradation, IR, and NMR spectroscopy analysis revealed that CFP-2 had a backbone consisting of α-(1→3) and α-(1→4)-linked d-glucopyranosyl residues substituted at O-6 with β-(1→6)-linked d-galactopyranosyl residue and α-(1→6)-linked d-mannopyranosyl residue. CFP-2 was able to reduce viability of cultured HL-60 and K562 cells. The antiproliferative properties of CFP-2 appeared to be attributable to its induction of apoptotic cell death as determined by ultrastructural change, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. To elucidate molecular events in the apoptosis, protein expressions of Bcl-2, Bax, Fas, and FasL were measured by Western blotting using specific antibodies in HL-60 cells. The level of Bcl-2 remained largely unchanged, but the Bax, Fas, and FasL expression showed up-regulation. Moreover, the telomerase activity analyzed by TRAP-ELISA assay in HL-60 cells treated with CFP-2 decreased as compared with the untreated control cells. These results suggest that CFP-2 could have a possible cancer therapeutic potential.
Keywords: Cladonia furcata; Lichenin; Apoptosis; Telomerase; HL-60 cell; K562 cell;

Urease enhances the formation of iron nitrosyl hemoglobin in the presence of hydroxyurea by Virginia L. Lockamy; Jinming Huang; Howard Shields; Samir K. Ballas; S.Bruce King; Daniel B. Kim-Shapiro (109-116).
Although it has been shown that hydroxyurea (HU) therapy produces measurable amounts of nitric oxide (NO) metabolites, including iron nitrosyl hemoglobin (HbNO) in patients with sickle cell disease, the in vivo mechanism for formation of these is not known. Much in vitro data and some in vivo data indicates that HU is the NO donor, but other studies suggest a role for nitric oxide synthase (NOS). In this study, we confirm that the NO-forming reactions of HU with hemoglobin (Hb) or other blood constituents is too slow to account for NO production measured in vivo. We hypothesize that, in vivo, HU is partially metabolized to hydroxylamine (HA), which quickly reacts with Hb to form methemoglobin (metHb) and HbNO. We show that addition of urease, which converts HU to HA, to a mixture of blood and HU, greatly enhances HbNO formation.
Keywords: Sickle cell disease; Hydroxyurea; Hydroxylamine; Nitric oxide;

Cell growth selection system to detect extracellular and transmembrane protein interactions by David M. Urech; Peter Lichtlen; Alcide Barberis (117-127).
The interplay among extracellular and cell surface proteins, such as the interactions between ligands and receptors or between antigens and antibodies, is involved in a multitude of physiological and pathological phenomena. In the oxidizing milieu of the secretory pathway in eukaryotic cells, many extracellular proteins build disulfide bonds that significantly contribute to their correct folding and structural stability. Thus, conventional yeast two-hybrid interaction assays, which occur in the reducing intracellular environment, might not be adequate to detect extracellular protein–protein interactions. We have exploited the properties of yeast Ire1p, a type I endoplasmic reticulum (ER) membrane protein involved in the unfolded protein response (UPR) as a dimerization-activated receptor, to develop a novel system for the detection and study of interactions between extracellular and/or membrane proteins. In our system, named SCINEX-P (screening for interactions between extracellular proteins), proteins of interest were fused to truncated Ire1p so as to substitute its N-terminal lumenal domain (NLD). Specific interaction between two partners caused dimerization of the Ire1p moiety, which, through the endogenous UPR signalling pathway, led to activation of transcription of genes that permit cell growth under selective conditions.
Keywords: Extracellular protein; Transmembrane protein; Antibody; Ligand; Receptor; Protein–protein interaction;

Triosephosphates are toxic to superoxide dismutase-deficient Escherichia coli by Ludmil Benov; Anees F. Beema; Fatima Sequeira (128-132).
Increase in the production of triosephosphates has been considered an important factor leading to diabetic complications. It might be expected that like the other short chain monosaccharides, triosephosphates autoxidize producing superoxide radical and α,β-diketones. Since superoxide can also initiate the oxidation of short chain sugars, free radical chain reactions are possible. If such reactions occur in vivo, triosephosphates would be more deleterious to cells lacking superoxide dismutase (SOD) than to normal cells. Here we demonstrate that triosephosphates kill a SOD-deficient Escherichia coli mutant much more than the parental, SOD-proficient strain. The effect is oxygen-dependent and is partially suppressed by aminoguanidine. Increased production of superoxide and diketones appeared to be the cause of triosephosphates toxicity.
Keywords: Dihydroxyacetone phosphate; d-Glyceraldehyde-3-phospate; Superoxide dismutase; Triosephosphates autoxidation; Methylglyoxal; Aminoguanidine;

Reduction of CMP-N-acetylneuraminic acid hydroxylase activity in engineered Chinese hamster ovary cells using an antisense-RNA strategy by Stephane Chenu; Anne Grégoire; Yanina Malykh; Athanase Visvikis; Lucia Monaco; Lee Shaw; Roland Schauer; Annie Marc; Jean-Louis Goergen (133-144).
Rodent cells, widely used for the industrial production of recombinant human glycoproteins, possess CMP-N-acetylneuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase; EC 1.14.13.45) which is the key enzyme in the formation of the sialic acid, N-glycolylneuraminic acid (Neu5Gc). This enzyme is not expressed in an active form in man and evidence suggests that the presence of Neu5Gc in recombinant therapeutic glycoproteins may elicit an immune response. The aim of this work was, therefore, to reduce CMP-Neu5Ac hydroxylase activity in a Chinese Hamster Ovary (CHO) cell line, and thus the Neu5Gc content of the resulting glycoconjugates, using a rational antisense RNA approach. For this purpose, the cDNA of the hamster hydroxylase was partially cloned and sequenced. Based on the sequence of the mouse and hamster cDNAs, optimal antisense RNA fragments were selected from preliminary in vitro translation tests. Compared to the parental cell line, the new strain (CHO-AsUH2), which was transfected with a 199-bp antisense fragment derived from the mouse CMP-Neu5Ac hydroxylase cDNA, showed an 80% reduction in hydroxylase activity. An analysis of the sialic acids present in the cells' own glycoconjugates revealed a decrease in the percentage of Neu5Gc residues from 4% in the parental cells to less than 1% in the CHO-AsUH2 cell line.
Keywords: CMP-Neu5Ac hydroxylase; Sialylation; N-glycolylneuraminic acid; CHO cell; Antisense RNA;