BBA - General Subjects (v.1780, #1)

Characterization of emY162 encoding an immunogenic protein cloned from an adult worm-specific cDNA library of Echinococcus multilocularis by Yoshinobu Katoh; Hirokazu Kouguchi; Jun Matsumoto; Akiko Goto; Tomohiro Suzuki; Yuzaburo Oku; Kinpei Yagi (1-6).
A cDNA library based on mRNA from adult worms of Echinococcus multilocularis was constructed. One cDNA clone, emY162, was isolated from this cDNA library. The putative protein from emY162 cDNA consists of 153 amino acids and has a predicted molecular weight of 17.0 kDa. The amino acid sequences of EMY162 are predicted to be a hydrophobic N-terminus conserving a secretory signal, and a hydrophobic C-terminus encoding a transmembrane domain or glycosyl-phosphatylinositol membrane anchor, and to have single fibronectin type III-like domain. In addition, it was shown that the emY162 gene (1738 bp) in the E. multilocularis genome DNA consists of three exons and two introns, and that emY162 is expressed in all four stages (protoscoleces, cultured metacestodes, immature adult worms and mature adult worms). Moreover, immunity to recombinant EMY162, which comprises the fibronectin type III-like domain on the EMY162 protein, was examined. Immune responses to the recombinant EMY162 were studied by using serum from dogs infected with E. multilocularis. Strong IgG immune responses were detected in Western blots.
Keywords: Echinococcus multilocularis; Adult worm; cDNA library; emY162 cDNA; Immunogenic protein; Recombinant EMY162 antigen;

Multi-block poloxamer surfactants suppress aggregation of denatured proteins by Devkumar Mustafi; Catherine M. Smith; Marvin W. Makinen; Raphael C. Lee (7-15).
On the basis of elastic light scattering, we have compared the capacity of the multi-block, surfactant copolymers Poloxamer 108 (P108), Poloxamer 188 (P188), and Tetronic 1107 (T1107), of average molecular weight 4700, 8400, and 15,000, respectively, with that of polyethylene glycol (PEG, molecular weight 8000) to suppress aggregation of heat-denatured hen egg white lysozyme (HEWL) and bovine serum albumin (BSA). We also compared the capacity of P188 to that of PEG to suppress aggregation of carboxypeptidase A denatured in the presence of trifluoroethanol and to facilitate recovery of catalytic activity. In contrast to the multi-block copolymers, PEG had no effect in inhibiting aggregation of HEWL or of carboxypeptidase A with the recovery of catalytic activity. At very high polymer:protein ratios (≥ 10:1), PEG increased aggregation of heat-denatured HEWL and BSA, consistent with its known properties to promote macromolecular crowding and crystallization of proteins. At a polymer:protein ratio of 2:1, the tetra-block copolymer T1107 was the most effective of the three surfactant copolymers, completely suppressing aggregation of heat-denatured HEWL. At a T1107:BSA ratio of 10:1, the poloxamer suppressed aggregation of heat-denatured BSA by 50% compared to that observed in the absence of the polymer. We showed that the extent of suppression of aggregation of heat-denatured proteins by multi-block surfactant copolymers is dependent on the size of the protein and the copolymer:protein molar ratio. We also concluded that at least one of the tertiary nitrogens in the ethylene-1,2-diamine structural core of the T1107 copolymer is protonated, and that this electrostatic factor underlies its capacity to suppress aggregation of denatured proteins more effectively than nonionic, multi-block poloxamers. These results indicate that amphiphilic, surfactant, multi-block copolymers are efficient as additives to suppress aggregation and to facilitate refolding of denatured proteins in solution. Because of these properties, multi-block, surfactant copolymers are suitable for application to a variety of biotechnological and biomedical problems in which refolding of denatured or misfolded proteins and suppression of aggregation are important objectives.
Keywords: Carboxypeptidase A; Light scattering; Lysozyme; Multi-block surfactant copolymer; Poloxamer; Protein denaturation; Serum albumin;

Differential effect of creatine on oxidatively-injured mitochondrial and nuclear DNA by Chiara Guidi; Lucia Potenza; Piero Sestili; Chiara Martinelli; Michele Guescini; Laura Stocchi; Sabrina Zeppa; Emanuela Polidori; Giosuè Annibalini; Vilberto Stocchi (16-26).
Creatine is a naturally occurring compound obtained in humans from endogenous production and consumption through the diet. It is used as an ergogenic aid to improve exercise performance and increase fat-free mass. Lately, creatine’s positive therapeutic benefits in various oxidative stress-associated diseases have been reported in literature and, more recently, creatine has also been shown to exert direct antioxidant effects. Oxidatively-challenged DNA was analysed to show possible protective effects of creatine. Acellular and cellular studies were carried out. Acellular assays, performed using molecular approaches, showed that creatine protects circular and linear DNA from oxidative attacks.Nuclear and mitochondrial DNAs from oxidatively-injured human umbilical vein endothelial cells were analyzed. Creatine supplementation showed significant genoprotective activity on mitochondrial DNA. This evidence suggests that creatine may play an important role in mitochondrial genome stability in that it could normalize mitochondrial mutagenesis and its functional consequences. Thus, creatine supplementation could be used to prevent or ameliorate diseases related to mitochondrial DNA mutations, and possibly to delay aging.
Keywords: Creatine; Oxidative DNA damage; Mitochondrial DNA; Nuclear DNA; Antioxidant;

A single nucleotide polymorphism in the human COMT (catechol-O-methyltransferase) gene has been associated with increased risk for breast cancer and several CNS diseases and disorders. The G to A polymorphism causes a valine (val) to methionine (met) substitution at codon 108 soluble - (S)/158 membrane - (MB)-COMT, generating alleles encoding high and low-activity forms of the enzyme, COMTH and COMTL, respectively. Tissues and cells with a COMTLL genotype have decreased COMT activity compared to COMTHH cells. Previously, we reported that the decreased activity was due to decreased amounts of S-COMTL protein in human hepatocytes. In this study, we investigated the role of S-COMT protein synthesis and turnover as determinates of reduced COMT protein in COMTLL compared to COMTHH cells. No association between S-COMT protein synthesis and COMT genotype was detected. Using a pulse-chase protocol, the half-life of S-COMTH was determined to be 4.7 days, which was considerably longer than expected from the half-lives of other phase 2 enzyme proteins. The half-life of S-COMTL compared to S-COMTH protein was significantly shorter at 3.0 days, but the difference was affected by the medium used during the chase period. These results suggest that increased turnover may contribute to reduced COMT activity in cells and tissues from COMTLL individuals. Subtle differences appear to be able to affect the stability of the S-COMTL protein, and this may contribute to the differences observed in epidemiological studies on the association of this polymorphism with breast cancer risk.
Keywords: Catechol-O-methyltransferase (COMT); COMT polymorphism; Protein synthesis; Protein turnover; Human breast epithelial cell lines;

Tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including alcoholic liver disease.  In the liver, Kupffer cells are the primary source of the cytokine. Obliteration of Kupffer cells or neutralization of TNF-α by anti-TNF-α antibody or by an antisense oligonucleotide prevents ethanol-mediated liver damage. In this study, we report the identification of yet another highly efficacious gene-silencing molecule, the short interfering RNA (siRNA), SSL3, against TNF-α. The efficacies of various siRNA duplexes were tested against TNF-α mRNA in primary cultures of rat Kupffer cells. SSL3 (25 nM) inhibited lipopolysaccharide (LPS)-induced secretion of TNF-α by 55% (p  < 0.005) with a proportionate reduction in TNF-α mRNA; the inhibitory effect lasted for at least 96 h. Four nucleotide mismatches to SSL3 completely abolished the inhibitory effects of SSL3, suggesting the sequence specificity of the siRNA. Further, the in vivo efficacy of SSL3 was assessed following the i.v. administration of two doses (140 μg/kg body weight/day for 2 days) of liposome-encapsulated SSL3. The LPS-induced TNF-α secretion was inhibited by > 60% (p  < 0.05) by SSL3 pre-treatment. These data demonstrate the identification of an siRNA against TNF-α, which, as a liposomal formulation, has therapeutic potential in the treatment of inflammatory diseases mediated by TNF-α.
Keywords: TNF alpha; siRNA; Kupffer cells; Lipopolysaccharide; Liposomes;

Water structure in vitro and within Saccharomyces cerevisiae yeast cells under conditions of heat shock by Jennifer L. Dashnau; Laura K. Conlin; Hillary C.M. Nelson; Jane M. Vanderkooi (41-50).
The OH stretch mode from water and organic hydroxyl groups have strong infrared absorption, the position of the band going to lower frequency with increased H-bonding. This band was used to study water in trehalose and glycerol solutions and in genetically modified yeast cells containing varying amounts of trehalose. Concentration-dependent changes in water structure induced by trehalose and glycerol in solution were detected, consistent with an increase of lower-energy H-bonds and interactions at the expense of higher-energy interactions. This result suggests that these molecules disrupt the water H-bond network in such a way as to strengthen molecule–water interactions while perturbing water–water interactions. The molecule-induced changes in the water H-bond network seen in solution do not translate to observable differences in yeast cells that are trehalose-deficient and trehalose-rich. Although comparison of yeast with low and high trehalose showed no observable effect on intracellular water structure, the structure of water in cells is different from that in bulk water. Cellular water exhibits a larger preference for lower-energy H-bonds or interactions over higher-energy interactions relative to that shown in bulk water. This effect is likely the result of the high concentration of biological molecules present in the cell. The ability of water to interact directly with polar groups on biological molecules may cause the preference seen for lower-energy interactions.
Keywords: Infrared; Heat shock; Hydrogen bond; Trehalose; Glycerol; Saccharomyces cerevisiae;

The objective of the present study was to isolate a lectin from fresh fruiting bodies of the mushroom Pleurotus citrinopileatus and examine it for various biological activities. The isolation procedure comprised ion exchange chromatography on DEAE-cellulose, CM-celluloses, and Q-Sepharose, and gel filtration on Superdex 75. A homodimeric 32.4 kDa lectin displaying high hemagglutinating activity was isolated with over 110 fold of purification. Its N-terminal amino acid sequence, QYSQMAQVME, has not been reported for other lectins. The lectin was unadsorbed on DEAE-cellulose in 0.001 M NH4HCO3 buffer (pH 9.4), but adsorbed on CM-cellulose in 0.001 M NH4OAc buffer (pH 4.8) and eluted by approximately 0.05 M NaCl in the same buffer. The lectin was subsequently adsorbed on Q-Sepharose and eluted by a linear gradient of 0–0.2 M NaCl in 10 mM NH4HCO3 buffer (pH 8.5). The lectin was obtained in a purified form after gel filtration by fast protein liquid chromatography on Superdex 75. The hemagglutinating activity of the lectin was inhibited by maltose, O-nitrophenyl-β-d-galactopyranoside, O/P-nitrophenyl-β-d-glucuronide and insulin. It was stable at temperatures up to 60 °C, and in NaOH and HCl solutions up to 0.1 M and 0.006 M concentration, respectively. It was sensitive to inhibition by HgCl2 and potentiation by AlCl3. The lectin exerted potent antitumor activity in mice bearing sarcoma 180, and caused approximately 80% inhibition of tumor growth when administered intraperitonealy at 5 mg/kg daily for 20 days. It elicited a mitogenic response from murine splenocytes in vitro with the maximal response at a lectin concentration of 2 μM. The lectin inhibited HIV-1 reverse transcriptase with an IC50 of 0.93 μM. It was devoid of antifungal activity.
Keywords: Lectin; Pleurotus citrinopileatus; Hemagglutinating activity; Antitumor activity; Mitogenic activity; HIV-1 reverse transcriptase inhibitory activity;

Nitroxides are more efficient inhibitors of oxidative damage to calf skin collagen than antioxidant vitamins by Elisabetta Venditti; Andrea Scirè; Fabio Tanfani; Lucedio Greci; Elisabetta Damiani (58-68).
Reactive oxygen species generated upon UV-A exposure appear to play a major role in dermal connective tissue transformations including degradation of skin collagen. Here we investigate on oxidative damage to collagen achieved by exposure to (i) UV-A irradiation and to (ii) AAPH-derived radicals and on its possible prevention using synthetic and natural antioxidants. Oxidative damage was identified through SDS-PAGE, circular dichroism spectroscopy and quantification of protein carbonyl residues. Collagen (2 mg/ml) exposed to UV-A and to AAPH-derived radicals was degraded in a time- and dose-dependent manner. Upon UV-A exposure, maximum damage was observable at 730 kJ/m2 UV-A, found to be equivalent to roughly 2 h of sunshine, while exposure to 5 mM AAPH for 2 h at 50 °C lead to maximum collagen degradation. In both cases, dose-dependent protection was achieved by incubation with μM concentrations of nitroxide radicals, where the extent of protection was shown to be dictated by their structural differences whereas the vitamins E and C proved less efficient inhibitors of collagen damage. These results suggest that nitroxide radicals may be able to prevent oxidative injury to dermal tissues in vivo alternatively to commonly used natural antioxidants.
Keywords: Collagen; SDS-PAGE; UV-A; Alkyl and peroxyl radicals; Carbonyl groups; Nitroxide antioxidants;