BBA - General Subjects (v.1570, #3)

Transforming growth factor β1 (TGFβ1) plays important roles in the regulation of cell growth and differentiation in both normal and malignant prostate epithelial cells. Although certain pathways have been suggested, the mechanisms responsible for the action of TGFβ1 are not well understood. In the present study, using a human papilloma virus 16 E6/E7 immortalized prostate epithelial cell line, HPr-1, we report that TGFβ1 was able to suppress the expression of Id-1, a helix–loop–helix (HLH) protein, which plays important roles in the inhibition of cell differentiation and growth arrest. In addition, a decrease at both Id-1 mRNA and protein expression levels was associated with TGFβ1-induced growth arrest and differentiation, indicating that Id-1 may be involved in TGFβ1 signaling pathway. The fact that up-regulation of p21WAF1, one of the downstream effectors of Id-1, was observed after exposure to TGFβ1 further indicates the involvement of Id-1 in the TGFβ1-induced growth arrest in HPr-1 cells. However, increased expression of p27KIP1 was also observed in the TGFβ1-treated cells, suggesting that in addition to down-regulation of Id-1, other factors may be involved in the TGFβ1-induced cell growth arrest and differentiation in prostate epithelial cells. Our results provide evidence for the first time that TGFβ1 may be one of the upstream regulators of Id-1.
Keywords: Prostate epithelial cell; TGFβ1; Id-1;

Pleurotus and Agrocybe hemolysins, new proteins hypothetically involved in fungal fruiting by Sabina Berne; Igor Križaj; Franc Pohleven; Tom Turk; Peter Maček; Kristina Sepčić (153-159).
Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg2+ but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.
Keywords: Pleurotus ostreatus; Agrocybe aegerita; Mushroom; Hemolytic protein; Fungal fructification;

Production and characterization of murine monoclonal anti-human DNase II antibodies, and their use for immunoaffinity purification of DNase II from human liver and urine by Tamiko Nakajima; Toshihiro Yasuda; Haruo Takeshita; Shinjiro Mori; Kouichi Mogi; Yasushi Kaneko; Emiko Nakazato; Koichiro Kishi (160-164).
Four murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion (SRED) and DNA-cast polyacrylamide gel electrophoresis (DNA-cast PAGE) were very useful for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining of the 32-kDa enzyme band, which is one of the three non-identical subunits of human DNase II molecule separated by sodium dodecyl sulfate (SDS)-PAGE followed by blotting on a transfer membrane. A formyl-cellulofine resin conjugated with each antibody specifically adsorbed and efficiently desorbed the active DNase II enzyme. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.
Keywords: Chromatography; DNase II; Human; Immunoaffinity; Liver; Monoclonal antibody; Murine; Purification; Urine;

The protection provided by poly(ethyleneimine) (PEI) to muscle lactate dehydrogenase (LDH) in metal-catalyzed oxidation (MCO) systems (CuSO4 or FeCl2 combined with H2O2) was studied, and comparisons were made with the chelators EDTA and desferal, respectively. The analytical chelating capacity of PEI was estimated to be around 1 mol Cu2+/10 mol ethyleneimine for all molecular weights of the polymer. The effect of [PEI monomer]/[metal ion] molar ratio on the oxidatively induced aggregation of LDH exhibited a similar trend as that of the other chelators; aggregation was enhanced at lower ratios and subsequently decreased until it was undetectable with increasing ratio. In contrast, the LDH activity showed a monotonic increase with increasing concentrations of the chelator. Total protection to the enzyme by PEI was provided at concentrations lower than that needed for full chelation of the copper ions, i.e. at [PEI monomer]/[Cu2+] ratio above 9 in case of PEI 2000, and above 7 for PEI 25 000 and 2.6×106, respectively. The polymer also provided protection against oxidation in an iron-based MCO system. Hydroxyl radical formation during the MCO reaction was inhibited in the presence of PEI. The polymer of higher molecular weights also exhibited a stronger free radical scavenging effect.
Keywords: Protein stabilization; Metal-catalyzed oxidation; Aggregation; Poly(ethyleneimine); Lactate dehydrogenase; Free radical;

Enzymatic properties, crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans by Yoshihiro Hakamada; Keiji Endo; Shuichi Takizawa; Tohru Kobayashi; Tsuyoshi Shirai; Takashi Yamane; Susumu Ito (174-180).
A high-isoelectric-point (pI), alkaline endo-1,4-β-glucanase (Egl-257) of Bacillus circulans KSM-N257 was purified to homogeneity and crystallized. The purified enzyme hydrolyzed carboxymethyl cellulose (CMC) with optima of pH 8.5 and 55 °C. The molecular mass was 43 kDa, and the pI was pH 9.3. The structural gene contained a single open reading frame of 1221 bp, corresponding to 407 amino acids (aa), including a 30-aa signal peptide (377 aa and 41,680 Da for the mature enzyme). Egl-257 hydrolyzed lichenan and showed 76.3% aa identity to a lichenase from B. circulans WL-12 belonging to glycosyl hydrolase family 8 but did not hydrolyze laminarin, curdran, and xylan at all. This indicates that Egl-257 is a true endo-1,4-β-glucanase. However, this enzyme was not active on p-nitrophenyl β-d-cellotrioside and p-nitrophenyl β-d-cellotetraoside. It was crystallized by the hanging-drop vapor-diffusion method with phosphate plus CdCl2 as precipitant. Pyramid-like crystals were formed, and they diffracted X-rays beyond 2.2 Å resolution. It belongs to the space group P212121 with unit cell parameters of a=62.5 Å, b=71.7 Å, and c=88.6 Å.
Keywords: Cellulase; Glycosyl hydrolase; Family 8; Cloning; Crystallization; Lichenase; Bacillus;

Rhus verniciflua Stokes (RVS) is a widely used herbal plant with various biological properties. Our previous study using cultured neuronal cells showed that an ethanol extract of RVS had strong antioxidant properties. In this study, we characterized the antioxidant activity of the RVS ethanol extract and identified the active compounds responsible for this activity. From the RVS ethanol extract, we derived three water-eluted fractions and another three fractions eluted by organic solvents, and determined that the water-eluted fractions are what protect against reactive oxygen species (ROS) generated by iron and enzymes. Water-eluted fraction F2 was the most efficient antioxidant. Moreover, DNA fragmentation and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining experiments revealed that F2 also protects against thymocyte apoptosis mediated by hydroxyl radicals. Finally, EI-MS, 1H-NMR, and 13C-NMR spectra signals confirmed that the fraction contained flavonoid derivatives, including fustin, quercetin, butein, and sulfuretin. These results suggest that the flavonoid derivatives in F2 are the compounds in the RVS ethanol extract that act as antioxidants.
Keywords: Antioxidant; Apoptosis; Flavonoid; Reactive oxygen specie; Rhus verniciflua Stokes;

Antimony(V) complex formation with adenine nucleosides in aqueous solution by Cynthia Demicheli; Frédéric Frézard; Marc Lecouvey; Arlette Garnier-Suillerot (192-198).
Despite the clinical use of pentavalent antimonial drugs for over half a century, their mode of action against leishmaniasis remains poorly understood. In this paper, we investigated the ability of Sb(V) to form in aqueous solution complexes with adenine nucleosides and deoxynucleosides, using circular dichroism (CD) and 1H and 13C NMR spectroscopies. We report that the ribonucleosides, adenosine (A) and adenosine monophosphate (AMP), form in water complexes with Sb(V), as evidenced by the changes induced in their CD spectra. On the other hand, 2′-deoxyadenosine (dA) did not show such a change. CD titration of the ribonucleosides with Sb(V) suggests the formation of 1:2 Sb(V)–nucleoside complexes. NMR analysis indicates that Sb(V) binds to the sugar moiety at the 2′ position. Furthermore, the incubation of the antimonial drug, meglumine antimonate, with adenosine at 37 °C led to the transfer of Sb(V) from its original ligand to the nucleoside molecule, at acidic pH (pH 5), but not at neutral pH (7.2). Our data therefore suggests that the formation of such complexes may take place in vivo within the acidic cell compartments, including the phagolysosome of macrophage in which Leishmania resides.
Keywords: Nucleoside; Adenosine; Antimony; Meglumine antimonate; Mechanism of action; Leishmaniasis;

Effects of the absence of Cu,Zn-superoxide dismutase (CuZnSOD) on the replicative life span of the yeast Saccharomyces cerevisiae were studied under different oxygen conditions. In both strains, replicative life span and the rate of cell divisions were found to be similar under the atmosphere of air and under hypoxic (3% oxygen) and anoxic conditions. These results indicate that deleterious consequences of the lack of CuZnSOD are not limited to elevation of superoxide concentration and involve function(s) other than superoxide scavenging.
Keywords: Aging; Yeast; Saccharomyces cerevisiae; Superoxide dismutase; Hypoxia; Anoxia;

Serum mannan binding protein inhibits mannosylated liposome-mediated transfection to macrophages by P Opanasopit; K Hyoudou; M Nishikawa; F Yamashita; M Hashida (203-209).
The effects of serum mannan binding proteins (MBP) in the transfection of plasmid DNA/Man–liposome complex via mannose receptor-mediated endocytosis was studied in vitro using cultured mouse peritoneal macrophages. Plasmid DNA encoding luciferase gene was complexed with cationic mannosylated liposomes (Man–liposomes), composed of cholesten-5-yloxy-N-(4-((1-imino-2-d-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) and dioleoyl phosphatidylethanolamine (DOPE). The transfection efficiency, as well as the binding and uptake of the plasmid DNA/Man–liposome complex, was investigated with or without serum MBP. The in vitro transfection efficiency of the complex was significantly reduced on increasing the amount of serum MBP. In addition, the cellular association of the complex was also reduced. These results indicate that serum MBP specifically binds to the mannose moieties on the complex and suppresses its cellular uptake, resulting in inhibition of the gene transfection in macrophages. Such an interaction is an obstacle to mannose receptor-mediated in vivo gene transfer to mannose receptor-positive cells using mannosylated gene carriers.
Keywords: Cationic liposome; Gene transfer; Mannose receptor; Mannan binding protein; Peritoneal macrophage;

The role of the cell cycle on the efficiency of photochemical gene transfection by Lina Prasmickaite; Anders Høgset; Kristian Berg (210-218).
The efficiency of gene transfection mediated by nonviral vectors is limited because of nonoptimal intracellular trafficking of transfecting DNA. Most nonviral vectors deliver transfecting DNA into a cell through endocytosis. However, poor escape from endocytic vesicles and inefficient transport of DNA into the nucleus often limits a success of gene transfection. Photochemical transfection is a new method, based on light-induced permeabilisation of endocytic vesicles, liberating transfecting DNA into the cytosol, concurrently increasing the chances for DNA to enter the nucleus.The aim of this study was to investigate the role of the cell cycle for the efficiency of photochemical transfection. It was demonstrated that in asynchronous human colon carcinoma HCT 116 cells photochemical treatment increased the transfection mediated by the nonviral vectors, the cationic polypeptide polylysine and the cationic lipid N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide/dioleoylphosphatidylethanolamine (βAE-DMRIE/DOPE), by 30- and 2.5-fold, respectively. In aphidicolin-synchronised cells, photochemical transfection mediated by polylysine was dependent on the cell cycle: transfection level was 4-fold higher when illumination, inducing photochemical reactions, was performed during the G2/M phase as compared to the G1/early-S phase. The cell cycle influence on photochemical transfection mediated by βAE-DMRIE/DOPE was very low: only 20% difference between G2/M and the G1/S phase was observed. We suggest that transgenes, photochemically liberated close/during mitosis, perhaps have the highest opportunity to enter the nucleus and be expressed. However, the dependence of photochemical transfection on the cell cycle might be partially disguised by various factors induced by photochemical treatment.
Keywords: Cell cycle; Gene transfection; Photochemical treatment; Endocytic vesicle; Mitosis;