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BBA - General Subjects (v.1770, #10)
Characterization of anti-coagulant properties of prenylated coumarin ferulenol by Monia Monti; Mirko Pinotti; Giovanni Appendino; Franco Dallocchio; Tiziana Bellini; Fabiana Antognoni; Ferruccio Poli; Francesco Bernardi (pp. 1437-1440).
We investigated the mechanisms underlying severe bleeding occurring upon consumption of Ferula communis. The prenylated coumarin ferulenol extracted from this plant did not directly affect blood coagulation but showed hepatocyte cytotoxicity and, at non-cytotoxic concentrations (<100 nM), impaired factor X biosynthesis (40% reduction). Studies with ferulenol derivatives indicated the prenyl residue as major determinant of ferulenol activity.
Keywords: Blood coagulation; Factor X; Ferulenol; Haemorrhagic tendency; Cellular models
Inhibition of L-selectin binding by polyacrylamide-based conjugates under defined flow conditions by Sven Enders; Gesche Bernhard; Andreas Zakrzewicz; Rudolf Tauber (pp. 1441-1449).
Selectins mediate tethering and rolling of leukocytes along the endothelium in a shear force-dependent manner. This key step in the cellular immune response is a target for experimental anti-inflammatory therapies. In the present paper we have examined the inhibitory activity of the minimal selectin ligand sialyl Lewis x (SiaLex), its isomer sialyl Lewis a (SiaLea) and sulfated tyrosine (sTyr) residues under dynamic flow reflecting the rheological conditions in the blood stream. The monomeric ligands were compared to multivalent polyacrylamide (PAA)-based conjugates under defined flow conditions on the molecular level, using surface plasmon resonance (SPR) technology, and on the cellular level, using a parallel-plate flow chamber. SPR measurements showed that a spatial arrangement of binding epitopes mimicking the selectin binding motif of the natural ligand PSGL-1 inhibits L-selectin binding successfully with IC50 values in the nanomolar range. Using a flow chamber adhesion assay it could be shown that the multivalent inhibitors efficiently blocked rolling and tethering of NALM-6 pre-B cells transfected with human L-selectin to activated endothelium and that the inhibitory activity increased with rising shear stress. While PAA-conjugates were almost not inhibitory at low shear stress, NALM-6 cell rolling was nearly completely inhibited at high shear stress. The results indicate that multimeric conjugates of SiaLex, SiaLea and sTyr are highly effective inhibitors of L-selectin-mediated cell adhesion particularly under flow conditions. Consequently, SiaLex, SiaLea and/or sTyr on macromolecular carriers may be promising candidates for anti-inflammatory therapy.
Keywords: Abbreviations; PAA; polyacrylamide; SiaLe; x; sialyl Lewis x; SiaLe; a; sialyl Lewis a; sTyr; sulfated tyrosine; SPR; surface plasmon resonanceL-selectin; PAA-based conjugates; Flow conditions; Inhibition; SPR; Flow chamber
The oncogenic fusion protein Pax3–FKHR has a greater post-translational stability relative to Pax3 during early myogenesis by Patrick J. Miller; Andrew D. Hollenbach (pp. 1450-1458).
The childhood solid muscle tumor Alveolar Rhabdomyosarcoma (ARMS) is characterized by the t(2;13)(q35;q14) chromosomal translocation, which results in the fusion of two transcription factors important for myogenesis, Pax3 and FKHR (FOX01a). The effects of myogenic differentiation on the stability of FKHR have been well characterized. However, similar studies have yet to be performed on Pax3 or the oncogenic fusion protein Pax3–FKHR. Therefore, we demonstrate in the physiologically relevant mouse primary myoblast system that the expression of Pax3 decreases nearly 95% during the first 24 h of myogenic differentiation. In contrast, there is an aberrant persistence of expression of Pax3–FKHR during this same time period. These differences in protein expression levels do not result from changes on the transcriptional nor the translational level since we observed no concomitant decrease in the levels of Pax3 or Pax3–FKHR mRNA or in the ability of both proteins to be translated. Instead, a pulse-chase analysis determined that Pax3–FKHR has a half-life significantly greater than the half-life of wild type Pax3 demonstrating for the first time that Pax3–FKHR has greater post-translational protein stability relative to wild type Pax3 during early myogenic differentiation. Finally, the persistence of expression of Pax3–FKHR prevents the terminal differentiation of primary myoblasts demonstrating a biological consequence of its aberrant expression.
Keywords: Alveolar rhabdomyosarcoma; Pax3; FKHR; Pax3–FKHR; Myogenesis; Protein stability
Sperm nuclei glutathione peroxidases and their occurrence in animal species with cysteine-containing protamines by Holger Bertelsmann; Markus Kuehbacher; Gundolf Weseloh; Antonios Kyriakopoulos; Dietrich Behne (pp. 1459-1467).
The selenoenzyme sperm nuclei glutathione peroxidase (snGPx), also called the nuclear form of phospholipid hydroperoxide glutathione peroxidase (n-PHGPx), was found to be involved in the stabilization of condensed sperm chromatin, most likely by thiol to disulfide oxidation of the cysteine residues of the mammalian protamines, small nuclear basic proteins in the nuclei of sperm cells. By applying Acidic Urea-PAGE in combination with SDS-PAGE, snGPx with an apparent molecular mass of 34 kDa and a 24-kDa protein were purified from rat sperm nuclei. The 24-kDa protein was identified by means of mass spectrometry as a truncated form of snGPx produced by cleavage at the N-terminal end. After defined processing of spermatozoa and detergent treatment of the sperm nuclei fraction, snGPx and its truncated form were shown to be the only selenoproteins present in mature mammalian sperm nuclei. Both forms were found in mature rat and horse sperm nuclei but in man only snGPx was detected. In trout and chicken, species with sperm cells which likewise undergo chromatin condensation but do not contain cysteine in their protamines, the snGPx proteins were missing. This can be taken as an indirect proof of the function of snGPx to act as protamine cysteine thiol peroxidase in the mammalian species with cysteine-containing protamines.
Keywords: Chromatin stabilization; Selenium; Sperm nuclei glutathione peroxidase; Protamine thiol peroxidation
De- N-glycosylation or G82S mutation of RAGE sensitizes its interaction with advanced glycation endproducts by Mari Osawa; Yasuhiko Yamamoto; Seiichi Munesue; Naho Murakami; Shigeru Sakurai; Takuo Watanabe; Hideto Yonekura; Yasuko Uchigata; Yasuhiko Iwamoto; Hiroshi Yamamoto (pp. 1468-1474).
Interactions between advanced glycation endproducts (AGE) and the receptor for AGE (RAGE) have been implicated in the development of diabetic vascular complications. RAGE has two N-glycosylation sites in and near the AGE-binding domain, and G82S mutation in the second N-glycosylation motif was recently reported in human. In this study, we examined whether de- N-glycosylation or G82S of RAGE affect its ability to bind AGE and cellular response to AGE. Recombinant wild-type, de- N-glycosylation and G82S RAGE proteins were produced in COS-7 cells, purified and assayed for ligand-binding abilities. De- N-glycosylation at N81 and G82S mutation decreased Kd for glycolaldehyde-derived AGE to three orders of magnitude lower levels compared with wild-type. AGE-induced upregulation of VEGF mRNA was significantly augmented in endothelial cell-derived ECV304 cells expressing de- N-glycosylated and G82S RAGE when compared with wild-type expressor. Exposure to low glucose resulted in the appearance of RAGE proteins of deglycosylated size in wild-type RAGE-expressing cells and significantly enhanced glycolaldehyde-derived AGE-induced VEGF mRNA expression. De- N-glycosylation or G82S mutation of RAGE increases affinity for AGE ligands, and may sensitize cells or conditions with it to AGE.
Keywords: Abbreviations; AGE; advanced glycation endproducts; RAGE; receptor for AGE; esRAGE; endogenous secretory RAGE; VEGF; vascular endothelial growth factor; BSA; bovine serum albumin; RU; response units; Kd; dissociation constantAdvanced glycation endproducts (AGE); Receptor for AGE (RAGE); N; -glycosylation; Mutation
The CCAAT-binding factor CBF/NF-Y regulates the human acetylcholinesterase promoter activity during calcium ionophore A23187-induced cell apoptosis by Hui Zhu; Wei Gao; Yu-fang Shi; Xue-Jun Zhang (pp. 1475-1482).
We previously reported that the expression of acetylcholinesterase during A23187-induced apoptosis of HeLa cells is regulated by Ca2+ mobilization through the modulation of mRNA stability and acetylcholinesterase promoter activity. Transactivation of the human acetylcholinesterase promoter by A23187 was partially mediated by the distal CCAAT motif within the −1270 to −1248 fragment of the human acetylcholinesterase promoter, which was bound by the CCAAT binding factor (CBF/NF-Y). In the present study, we investigated the molecular mechanisms by which CBF/NF-Y regulates A23187-induced activation of the human acetylcholinesterase promoter. The results indicate that CBF/NF-Y binding to the distal CCAAT motif suppresses the promoter activity. Electrophoretic mobility shift assays (EMSAs) demonstrated that binding of CBF/NF-Y to the distal CCAAT motif decreased after A23187 treatment. Our results suggest that acetylcholinesterase promoter activation during A23187-induced HeLa cell apoptosis may result partly from the dissociation of CBF/NF-Y from the distal CCAAT motif in the acetylcholinesterase promoter, reversing this suppression.
Keywords: Abbreviations; AChE; Acetylcholinesterase acetylcholine acetyl hydrolase; CBF/NF-Y; the CCAAT-binding Factor; EMSAs; electrophoretic mobility shift assays; RNAi; RNA interference; PMSF; phenylmethylsulfonyl fluorideAcetylcholinesterase; CBF/NF-Y; Promoter activity; The distal CCAAT motif
Purification of the receptor for the N-acetyl-d-glucosamine specific adhesin of Mannheimia haemolytica from bovine neutrophils by Alfonso De la Mora; Francisco Suárez-Güemes; Francisco Trigo; Patricia Gorocica; Carlos Solórzano; Marie-Christine Slomianny; Concepción Agundis; M. Ali Pereyra; Edgar Zenteno (pp. 1483-1489).
The GlcNAc-specific adhesin from Mannheimia haemolytica (MhA) has been shown to participate in pathogenicity of mannheimiosis due to its capacity to adhere to tracheal epithelial cells and activate the oxidative burst of bovine neutrophils. In this work, we purified the MhA receptor from bovine neutrophils (MhAr) by affinity chromatography on MhA-Sepharose. The MhAr, which corresponded to approximately 2% of the protein from cell lysate, is a glycoprotein mainly composed of Glu, Ala, Ser, Gly, and Asp, without cysteine. The glycan portion, which corresponds to 20% by weight, is composed of GalNAc, GlcNAc, Man, Gal, and NeuAc. The receptor is a 165-kDa glycoprotein, as determined by molecular sieve chromatography under native conditions; SDS-PAGE analysis shows a heterodimer of 83 and 80 kDa subunits. This work suggests that the GlcNAc-containing receptor plays a relevant role by activating bovine neutrophils through non-opsonic mechanisms.
Keywords: Abbreviations; MhA; Purified adhesin from; Mannheimia haemolytica; NBT; nitroblue tetrazolium; PMA; phorbol 12-myristate 13-acetate; GalNAc; N-acetyl-; d; -galactosamine; GlcNAc; N-acetyl-; d; -glucosamine; NeuAc; sialic acid, N-acetyl-neuraminic acid; GlcNH; 2; d; -glucosamine Mannheimia haemolytica; Adhesin; GlcNAc-specificity; Bovine; Mannheimiosis; Neutrophils-oxidative burst
Establishment of the active catalytic domain of human PDGFRβ tyrosine kinase-based ELISA assay for inhibitor screening by Xiu-Hua Zhang; Xiao-Ning Guo; Li Zhong; Xiao-Min Luo; Hua-Liang Jiang; Li-Ping Lin; Jian Ding (pp. 1490-1497).
Tyrosine kinases are emerging as frequent targets of primary oncogenic events and therefore represent an optimal focus of therapeutic intervention. In an effort towards therapeutic PDGFR inactivation, we expressed the catalytic domain of PDGFRβ as a soluble active kinase using Bac-to-Bac expression system, and studied the correlations between PDGFRβ activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. And a convenient, effective and non-radioactive ELISA screening model is then established for identification of the potential inhibitors targeting PDGFRβ kinase. Of 500 RTK target-based compounds, TKI-30 was identified as a small molecule potential inhibitor of PDGFRβ (IC50=0.34 μM). Further studies indicated that TKI-30 blocked PDGF-BB-induced autophosphorylation of PDGFRβ in a dose-dependent manner in Swiss 3T3 cells and human umbilical vein smooth muscle cells (HUVSMCs). Moreover, it dose-dependently suppressed the PDGF-BB-induced proliferation in HUVSMCs and tube formation of HUVEC. Our data collectively indicated that PDGFRβ-based ELISA assay is a new method available for screening inhibitors targeting PDGFRβ kinase and TKI-30 is a potential novel anti-cancer agent worthy of being further investigated.
Keywords: Abbreviations; ATP; adenosine triphosphate; PDGF; Platelet-derived growth factor; RTK; receptor tyrosine kinase; DMSO; dimethyl sulfoxide; DTT; dithiothreitol; PMSF; phenylmethylsulfonyl fluoride; SDS; sodium dodecyl sulfate; PBS; phosphate-buffered saline; ELISA; enzyme-link-immunosorbent assay; HUVEC; human umbilical vein endothelial cell.PDGFRβ; Bac-to-Bac system; Tyrosine kinase inhibitors; TKI-30
Characterization of the adenine nucleoside specific phosphorylase of Bacillus cereus by Francesco Sgarrella; Luciano Frassetto; Simone Allegrini; Marcella Camici; Maria Caterina Carta; Paolo Fadda; Maria Grazia Tozzi; Piero Luigi Ipata (pp. 1498-1505).
Adenosine phosphorylase, a purine nucleoside phosphorylase endowed with high specificity for adenine nucleosides, was purified 117-fold from vegetative forms of Bacillus cereus. The purification procedure included ammonium sulphate fractionation, pH 4 treatment, ion exchange chromatography on DEAE-Sephacel, gel filtration on Sephacryl S-300 HR and affinity chromatography on N6-adenosyl agarose. The enzyme shows a good stability to both temperature and pH. It appears to be a homohexamer of 164±5 kDa. Kinetic characterization confirmed the specificity of this phosphorylase for 6-aminopurine nucleosides. Adenosine was the preferred substrate for nucleoside phosphorolysis ( kcat/ Km 2.1×106 s−1 M−1), followed by 2′-deoxyadenosine ( kcat/ Km 4.2×105 s−1 M−1). Apparently, the low specificity of adenosine phosphorylase towards 6-oxopurine nucleosides is due to a slow catalytic rate rather than to poor substrate binding.
Keywords: Adenosine phosphorylase; Purine nucleoside phosphorylase; Purine nucleosides; Bacillus cereus
Purification of β-galactosidase from Erythrina indica: Involvement of tryptophan in active site by Rakesh M. Kestwal; Shobhana V. Bhide (pp. 1506-1512).
β-Galactosidase (EC: 3.2.1.23), one of the glycosidases detected in Erythrina indica seeds, was purified to 135 fold. Amongst the four major glycosidases detected β-galactosidase was found to be least glycosylated, and was not retained by Con-A CL Seralose affinity matrix. A homogenous preparation of the enzyme was obtained by ion-exchange chromatography, followed by gel filtration. The enzyme was found to be a dimmer with a molecular weight of 74 kDa and 78 kDa, by gel filtration and SDS-PAGE, respectively. The optimum pH and optimum temperature for enzyme activity were 4.4 and 50 °C, respectively. The enzyme showed a Km value of 2.6 mM and Vmax of 3.86 U/mg for p-nitrophenyl-β-D-galactopyranoside as substrate and was inhibited by Zn2+ and Hg2+. The enzyme activity was regulated by feed back inhibition as it was found to be inhibited by β-D-galactose. Chemical modification studies revealed involvement of tryptophan and histidine for enzyme activity. Involvement of tryptophan was also supported by fluorescence studies and one tryptophan was found to be present in the active site of β-galactosidase. Circular dichroism studies revealed 37% α helix, 27% β sheet and 38% random coil in the secondary structure of the purified enzyme.
Keywords: Abbreviations; NBS; N-bromosuccinimide; Con-A; Concanavalin-A; PMSF; Phenyl methyl sulfonyl fluoride; DTNB; Dithio-bis 2-nitrobenzoicacid; PG; Phenyl glyoxal; NAI; N-acetyl imidazole; DEPC; Diethylpyro carbonate; DCCDI; N,N′; -Dicyclohexyl carbodiimide; CD; Circular dichroism Erythrina indica; β-galactosidase; Purification; CMC; Tryptophan