BBA - General Subjects (v.1770, #12)

An 18-kDa ribonuclease (RNase) with a novel N-terminal sequence was purified from fresh fruiting bodies of the mushroom Hypsizigus marmoreus. The purification protocol comprised ion exchange chromatography on DEAE cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose and Q-Sepharose and gel filtration by fast protein liquid chromatography on Superdex 75. The starting buffer was 10 mM Tris–HCl buffer (pH 7.2), 10 mM Tris–HCl buffer (pH 7.2), 10 mM NH4OAc buffer (pH 5), 10 mM NH4HCO3 buffer (pH 9.4) and 200 mM NH4HCO3 (pH 8.5), respectively. Absorbed proteins were desorbed using NaCl added to the starting buffer. A 42-fold purification of the enzyme was achieved. The RNase was unadsorbed on DEAE cellulose, Affi-gel blue gel and CM-cellulose but adsorbed on Q-Sepharose. It exhibited maximal RNase activity at pH 5 and 70 °C. Some RNase activity was detectable at 100 °C. It demonstrated the highest ribonucleolytic activity (196 U/mg) toward poly C, the next highest activity (126 U/mg) toward poly A, and much weaker activity toward poly U (48 U/mg) and poly G (41 U/mg). The RNase inhibited [3H-methyl]-thymidine uptake by leukemia L1210 cells with an IC50 of 60 μM.
Keywords: Ribonuclease; Antiproliferative; Mushroom; Hypsizigus marmoreus;

Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm (Bombyx mori) by Lingyin Kong; Zhengbing Lv; Jian Chen; Zuoming Nie; Dan Wang; Hongdan Shen; Xuedong Wang; Xiangfu Wu; Yaozhou Zhang (1598-1604).
14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3ζ and Bm14-3-3ε). Category of two 14-3-3 proteins was identified according to phylogenetic analysis. Bm14-3-3ζ shared 90% identity with that in Drosophila, while Bm14-3-3ε shared 86% identity with that in Drosophila. According to Western blot and real time PCR analysis, the Bm14-3-3ζ expression levels are higher than Bm14-3-3ε in seven tissues and in four silkworm developmental stages examined. Bm14-3-3ζ was expressed during every stage of silkworm and in every tissue of the fifth instar larvae that was examined, but Bm14-3-3ε expression was not detected in eggs or heads of the fifth instar larvae. Both 14-3-3 proteins were highly expressed in silk glands. These results suggest that Bm14-3-3ζ expression is universal and continuous, while Bm14-3-3ε expression is tissue and stage-specific. Based on tissue expression patterns and the known functions of 14-3-3 proteins, it may be that both 14-3-3 proteins are involved in the regulation of gene expression in silkworm silk glands.
Keywords: Bombyx mori; 14-3-3 protein; Expression analysis; EST; Real time PCR;

Trypanosoma cruzi, the etiologic agent of Chagas' disease, is a polyamine auxotroph organism because its genome contains neither ornithine decarboxylase (ODC) nor arginine decarboxylase (ADC) genes, presumably lost during evolution. After transformation with a recombinant plasmid bearing the complete coding region of Crithidia fasciculata ODC gene, the transgenic parasites were able to synthesize putrescine and simultaneously became susceptible to α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. We have studied the emergence of DFMO-resistant T. cruzi after one-step selection of ODC-transformed parasites cultivated in the presence of high levels of the drug (5 mM). Our results have indicated a duplication of the ODC gene copy number in the drug-resistant cell line. The ODC transcripts and the corresponding translation products showed very significant increases (about 7- and 25-fold, respectively) in DFMO-resistant parasites, while the ODC enzymatic activity was 5 times higher than in drug-sensitive T. cruzi. The unequal increases of ODC protein and enzymatic activity in DFMO-resistant protozoa strongly suggest that in addition to gene amplification and enhanced transcription and translation, the assembly of ODC polypeptide chains into dimeric active enzyme molecules might also contribute to regulate the development of DFMO resistance.
Keywords: Trypanosoma cruzi; Ornithine decarboxylase; Polyamine biosynthesis; DFMO resistance; Transgenic parasites;

Peroxisome proliferator-activated receptor gamma agonist action of 3-methyl-1,2-cyclopentanedione by Seo Young Choi; Jae Heun Chung; Dae Hyun Kim; Sang Woon Chung; Ji Young Kim; Byung Pal Yu; Hae Young Chung (1612-1619).
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that plays a pivotal role in regulating inflammatory gene expression. The purpose of this study was to investigate the effects of coffee extract, 3-methyl-1,2-cyclopentanedione (3-MCP) on PPARs in vitro. Western blotting and luciferase assays using the PPAR response element (PPRE) construct revealed that 3-MCP induced PPARγ-selective activation in YPEN-1 cells and that treatment with the PPARγ selective antagonist, GW9662, was associated with a decrease in 3-MCP-induced PPARγ activity. The 3-MCP also was shown to suppress reactive species generation and pro-inflammatory transcription factor NF-κB activity through PPARγ activation. Theses results indicate that 3-MCP is a novel PPARγ agonist and suggests that this agent may have a potential to minimize inflammation.
Keywords: 3-Methyl-1,2-cyclopentanedione; PPARγ agonist; Coffee; NF-κB; Inflammation;

Using giant unilamellar lipid vesicle micro-patterns as ultrasmall reaction containers to observe reversible ATP synthesis/hydrolysis of F0F1-ATPase directly by Xiaolong Liu; Rui Zhao; Yun Zhang; Xingyu Jiang; Jiachang Yue; Peidong Jiang; Zhenxi Zhang (1620-1626).
F0F1-ATPase within chromatophores, which was labeled with pH-sensitive quantum dots, was encapsulated in large unilamellar lipid vesicles (LUVs) through reverse-phase evaporation. Then a microarray of chromatophore-containing LUVs was created using a micro-contact printing (μ-CP) technique. Through controlled dehydration–rehydration of the lipid patterns, a microarray of single chromatophore-containing giant unilamellar lipid vesicles (GUVs) was formed with desired size and uniform shape. The reversible ATP synthesis/hydrolysis of F0F1-ATPase in GUVs was directly observed by fluorescence microscopy through the fluorescence intensity increase/decrease in the pH-sensitive quantum dots labeled on the outer surface of the chromatophore. To the best of our knowledge, this is the first direct observation of the reversible behavior of F0F1-ATPase at the bulk scale.
Keywords: F0F1-ATPase; Micro-contact printing (μ-CP); Giant unilamellar lipid vesicle (GUVs); Quantum dots; Chromatophore;

Plant collagenase: Unique collagenolytic activity of cysteine proteases from ginger by Misook Kim; Susan E. Hamilton; Luke W. Guddat; Christopher M. Overall (1627-1635).
Two cysteine proteases, GP2 and GP3, have been isolated from ginger rhizomes (Zingiber officinale). GP2 is virtually identical to a previously identified ginger protease GPII [K.H. Choi, and R.A. Laursen, Amino-acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale, Eur. J. Biochem. 267 (2000) 1516–1526.], and cleaves native type I collagen at multiple discrete sites, which are in the interior of the triple helical region of this molecule. In reaction with proline-containing peptides GP2 shows preference for Pro in the P2 position, and at least 10-fold higher efficiency of hydrolysis than papain. Comparison of models of GP2 and GP3 with the crystal structure of papain shows that the three enzymes have different S2 pocket structures. The S2 pocket in GP2 and GP3 is half the size of that of papain. GP2 is the only reported plant cysteine protease with a demonstrated ability to hydrolyse native collagen. The results support a role for ginger proteases as an alternative to papain, in commercial applications such as meat tenderization, where collagen is the target substrate.
Keywords: Plant; Ginger; Collagenase; Hydrolysis; Proline specificity; Meat tenderization;

A key step in the rational design of new RNA binding small molecules necessitates a complete elucidation of the molecular aspects of the binding of existing molecules to RNA structures. This work focuses towards the understanding of the interaction of a DNA intercalator, quinacrine and a minor groove binder 4′,6-diamidino-2-phenylindole (DAPI) with the right handed Watson–Crick base paired A-form and the left-handed Hoogsteen base paired HL-form of poly(rC)·poly(rG) evaluated by multifaceted spectroscopic and viscometric techniques. The energetics of their interaction has also been elucidated by isothermal titration calorimetry. Results of this study converge to suggest that (i) quinacrine intercalates to both A-form and HL-form of poly(rC)·poly(rG); (ii) DAPI shows both intercalative and groove-binding modes to the A-form of the RNA but binds by intercalative mode to the HL-form. Isothermal calorimetric patterns of quinacrine binding to both the forms of RNA and of DAPI binding to the HL-form are indicative of single binding while the binding of DAPI to the A-form reveals two kinds of binding. The binding of both the drugs to both conformations of RNA is exothermic; while the binding of quinacrine to both conformations and DAPI to the A-form (first site) is entropy driven, the binding of DAPI to the second site of A-form and HL-conformation is enthalpy driven. Temperature dependence of the binding enthalpy revealed that the RNA–ligand interaction reactions are accompanied by small heat capacity changes that are nonetheless significant. We conclude that the binding affinity characteristics and energetics of interaction of these DNA binding molecules to the RNA conformations are significantly different and may serve as data for the development of effective structure selective RNA-based antiviral drugs.
Keywords: A-form RNA; HL-form RNA; Quinacrine; DAPI; Intercalator; Groove binder; RNA–ligand interaction; Spectroscopy; Calorimetry; Thermodynamics;

Elevated cholesterol reduces acetylsalicylic acid-mediated platelet acetylation by Magdalena Boncler; Peter Gresner; Marek Nocun; Joanna Rywaniak; Martin Dolnik; Jacek Rysz; Radoslaw Wilk; Malgorzata Czyz; Leszek Markuszewski; Maciej Banach; Cezary Watala (1651-1659).
We describe the role of plasma and platelet cholesterol content in the ability of acetylsalicylic acid (ASA) to acetylate platelet proteins and inhibit platelet function. Platelet susceptibility to ASA was monitored in subjects differing in plasma total cholesterol and in suspensions of cholesterol-enriched or cholesterol-depleted platelets. Platelets from subjects with higher plasma cholesterol (> 6 mmol/l) showed reduced platelet sensitivity to ASA (inhibition of platelet aggregation and thromboxane generation by 60% and 68% in ‘lower-‘ vs. 32% and 56% in ‘higher-cholesterol’ donors; n  = 13 in each group; p  = 0.056 and p  < 0.04, respectively). [Acetyl-1-14C] incorporation to platelet proteins in subjects with higher plasma cholesterol was significantly reduced (11.0 vs. 14.6 nmol/g protein, p  < 0.0001) and correlated significantly with blood total cholesterolemia (R K  = − 0.430, p  < 0.003) and LDL-cholesterol (R K  = − 0.349, p  < 0.012), but not with platelet cholesterol content. In conclusion, elevated plasma cholesterol is an important determinant of ASA-induced acetylation of platelets and platelet diminished sensitivity to ASA. The molecular basis of such an association remains obscure, notwithstanding it may constitute a link between sub-optimal platelet response to aspirin and lipid metabolic disorders.
Keywords: Acetylsalicylic acid; Aspirin resistance; Cholesterol; Platelet aggregation; Platelet protein acetylation;

Endocytosis of pulchellin and its recombinant B-chain into K-562 cells: Binding and uptake studies by Leandro S. Goto; Priscila V. Castilho; Márcia R. Cominetti; Heloísa S. Selistre-Araújo; Ana Paula Ulian Araújo (1660-1666).
Most of the type 2 ribosome-inactivating proteins (RIPs) are toxins formed by an RNA–N-glycosidase A-chain polypeptide linked to a lectin B-chain by a single disulfide bond. Members of this protein class vary greatly in cytotoxity, correlating more with B-chain diversity rather than to A-chain differences. Pulchellin is a type 2 ribosome-inactivating protein toxin found in the seeds of Abrus pulchellus tenuiflorus. Recombinant pulchellin B-Chain (rPBC) has been previously produced as inclusion bodies in Escherichia coli and successfully refolded recovering biological activity. New approaches for using this kind of protein as a biotechnological tool require a better understanding of cell targeting, binding, uptake, intracellular routing and delivery. In this work, cell adhesion experiments were used to determine the interaction of rPBC with mammalian cells. Fluorescence and confocal microscopy revealed the intracellular localization and trafficking. Subcellular sorting of the native pulchellin could also be determined. The results support that the endosomal internalization pathway and the retrograde transport through the Golgi apparatus might be used by both native protein and rPBC.
Keywords: Ribosome-inactivating protein; Pulchellin; Endocytosis; Plant toxin;