Check out our New Publishers' Select for Free Articles

BBA - General Subjects (v.1760, #3)

Editorial Board (pp. ii).

Dictyostelium discoideum to human cells: Pharmacogenetic studies demonstrate a role for sphingolipids in chemoresistance by Stephen Alexander; Junxia Min; Hannah Alexander (pp. 301-309).

Resistance to chemotherapy is a major obstacle for the treatment of cancer and a subject of extensive research. Numerous mechanisms of drug resistance have been proposed, and they differ for different drugs. Nevertheless, it is clear that our understanding of this important problem is still incomplete, and that new targets for modulating therapy still await discovery. The attractive biology and the availability of powerful molecular techniques have made the cellular slime mold Dictyostelium discoideum, a powerful non-mammalian model for drug target discovery, and the problem of drug resistance. To understand the molecular basis of chemoresistance to the widely used drug cisplatin, both genetic and pharmacological approaches have been applied to this versatile experimental system. These studies have resulted in the identification of novel molecular pathways which can be used to increase the efficacy of cisplatin, and brought attention to the role of sphingolipids in mediating the cellular response to chemotherapeutic drugs. In the following review, we will describe the history and utility of D. discoideum in pharmacogenetics, and discuss recent studies which focus attention on the role of sphingolipids in chemotherapy and chemoresistance.

Keywords: Cancer therapy; Signal transduction modulator; MAP kinase; Cisplatin; Sphingosine 1-phosphate; Ceramide; Glutathione

Purification and characterization of an esterase isozyme involved in hydrolysis of organophosphorus compounds from an insecticide resistant pest, Helicoverpa armigera (Lepidoptera: Noct�idae) by R. Srinivas; S.K. Jayalakshmi; K. Sreeramulu; N.E. Sherman; Jayasimha Rao (pp. 310-317).

An esterase isozyme was purified from the insecticide resistant pest, Helicoverpa armigera collected from field crops. Purification involved ammonium sulfate precipitation, hydrophobic interaction and ion exchange chromatography followed by gel filtration chromatography. The purification was 212-fold with 1% yield of the enzyme. The optimum pH of the isozyme was found to be 10.5 and 8.5 for p-nitrophenyl phosphate and paraoxon, respectively. The enzyme was unstable at temperature >50 °C. The molecular mass determined by SDS-PAGE was 66 kDa. Cations such as Hg⁺², Ag⁺², Cd⁺² inhibited the activity while Zn⁺² stimulated it. Kinetic studies indicated that the enzyme had low K_m values of 0.238 and 0.348 mM for p-nitrophenyl phosphate and paraoxon, respectively. The enzyme had broad substrate specificity with high K_m values for ATP, ADP and β-glycerophosphate. This enzyme was partially sequenced and identified as an alkaline phosphatase.

Keywords: Helicoverpa armigera; Esterase; Paraoxon; Alkaline phosphatase; Sequence

Characterization of a recombinant C-type lectin, rCEL-IV, expressed in Escherichia coli cells using a synthetic gene by Tomomitsu Hatakeyama; Takao Hozawa; Iyo Hirotani; Nobuaki Tsuda; Masami Kusunoki; Kohei Shiba (pp. 318-325).

The body fluid of marine invertebrate Cucumaria echinata (Holothuroidea) contains four Ca²⁺-dependent galactose-specific lectins. One of these lectins, CEL-IV, is composed of a C-type carbohydrate-recognition domain homotetramer. CEL-IV exhibits higher specificity for α-galactosides than for β-galactosides, while other C. echinata lectins show preferential binding of β-galactosides. We constructed an artificial synthetic gene for recombinant CEL-IV (rCEL-IV) based on the amino acid sequence previously determined from the purified protein. rCEL-IV was expressed in Escherichia coli cells as inclusion bodies. After the refolding process, most of rCEL-IV spontaneously formed a homotetramer structure having interchain disulfide bonds. The secondary structure of rCEL-IV was similar to that of the native one, as judged by the comparison of the far UV-circular dichroism spectra of rCEL-IV and native CEL-IV (nCEL-IV). Carbohydrate-binding specificity of rCEL-IV was confirmed to be similar to that of nCEL-IV from the results of the binding-inhibition assay using liposomes composed of rabbit erythrocyte lipids. Crystals of rCEL-IV were obtained in a few days by the sitting drop vapor diffusion method. These results indicate that rCEL-IV achieved essentially correct three-dimensional structure, including the carbohydrate-binding sites, and it would be very useful for further study on the carbohydrate-recognition mechanism by mutational and X-ray crystallographic analyses.

Keywords: Abbreviations; CRD; carbohydrate-recognition domain; CTLD; C-type lectin-like domain; CD; circular dichroism; DLS; dynamic light scattering; EDTA; ethylenediamine tetraacetate; TBS; Tris-buffered saline; MALDI-TOF; matrix-assisted laser desorption ionization time-of-flight; rCEL-IV; recombinant CEL-IV; nCEL-IV; native CEL-IV; RE; rabbit erythrocyteArtificial gene; C-type lectin; Cucumaria echinata; Marine invertebrate; Expression; Crystal; Dynamic light scattering

Purification and characterization of an N-acetyl-d-galactosamine-specific lectin from the edible mushroom Schizophyllum commune by Podjana Chumkhunthod; Sureelak Rodtong; Stan J. Lambert; Anthony P. Fordham-Skelton; Pierre J. Rizkallah; Mark C. Wilkinson; Colin D. Reynolds (pp. 326-332).

An N-acetyl-d-galactosamine (GalNAc)-specific lectin was purified from the edible mushroom, Schizophyllum commune, using affinity chromatography on a porcine stomach mucin (PSM)-Sepharose 4B column. Under reducing and non-reducing conditions, SDS-polyacrylamide gel electrophoresis gave a major band of 31.5 kDa. The Schizophyllum commune lectin (SCL) showed high affinity toward rat erythrocytes and the sugar inhibition assay exhibited its sugar specificity highly toward lactose and N-acetyl-d-galactosamine. It was stable at 55 °C for 30 min and at pH 3–10 for 18-h test. The lectin was shown to be a glycoprotein with cytotoxic activity against human epidermoid carcinoma cells. The N-terminus of SCL was blocked but amino acid sequences of internal tryptic peptides showed moderately sequence similarities with some other fungal and plant lectins. Crystals of SCL were obtained by the sitting drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant, and gave an X-ray diffraction pattern to approximately 3.8 Å resolution.

Keywords: Lectin; Edible mushroom; N; -acetyl-; d; -galactosamine-specific lectin; Lectin crystal; Schizophyllum commune; X-ray diffraction

Interactions of hypocrellin B with hyaluronan and photo-induced interactions by Liming Song; Baozhong Zhao; Jie Xie; Jingquan Zhao (pp. 333-339).

In the current work, the molecular recognition and interaction were studied by taking advantages of the environmentally sensitive fluorescence of hypocrellin B (HB) and the structural knowledge of hyaluronan (HYA), a polysaccharide over-expressed in tumor cells or tissues. Interestingly, it was found that, binding to HYA, the absorbance of HB would be greatly strengthened, suggesting HB fitting to a hydrophobic environment in HYA, while the fluorescence seriously quenched at pH 7.0, which was very distinct from the binding of HB to proteins, liposome, other polysaccharide molecules or HYA at pH 2.0. Synchronously, the particle size of HYA would become bigger after interaction with HB, suggesting an aggregation of HYA. Considering the spectral responses of HB and the particle size change of HYA, a specific interaction of HB with HYA was proposed, that is, an HB molecule would link two HYA molecules not only by hydrophobic interaction but also by formations of intermolecular hydrogen bonds at physiological pH values. Furthermore, the estimated binding constant suggests a quite high affinity of HB to HYA. Besides, an oxygen-dependent degradation of HYA and photobleaching of HB were observed via photosensitization of HB.

Keywords: Abbreviations; HB; hypocrellin B; HYA; hyaluronan; PBS; Phosphate buffered salineEnvironmentally sensitive fluorescence of HB; Polysaccharides; HYA; Specific interaction; Intermolecular hydrogen bond

Trehalose protects Saccharomyces cerevisiae from lipid peroxidation during oxidative stress by R.S. Herdeiro; M.D. Pereira; A.D. Panek; E.C.A. Eleutherio (pp. 340-346).

Aiming to focus the protective role of the sugar trehalose under oxidative conditions, two sets of Saccharomyces cerevisiae strains, having different profiles of trehalose synthesis, were used. Cells were treated either with a 10% trehalose solution or with a heat treatment (which leads to trehalose accumulation) and then exposed either to menadione (a source of superoxide) or to tert-butylhydroperoxide (TBOOH). According to our results, trehalose markedly increased viability upon exposure to menadione stress, which seems to be correlated with decrease in lipid peroxidation levels. The protective effect of trehalose against oxidative damage produced by menadione was especially efficient under SOD1 deficiency. On the other hand, this sugar does not seem to participate of the mechanism of acquisition of tolerance against TBOOH, since trehalose pretreatment (addition of external trehalose) was not capable of increase cell survival. Therefore, trehalose plays a role in protecting cells, especially membranes, from oxidative injuries. However, this mechanism of defense is dependent on the type of oxidative stress to which cells are submitted.

Keywords: Trehalose; Menadione; Peroxide; Lipid peroxidation; Saccharomyces cerevisiae

Ovotransferrin is a redox-dependent autoprocessing protein incorporating four consensus self-cleaving motifs flanking the two kringles by Hisham Radwan Ibrahim; Taku Haraguchi; Takayoshi Aoki (pp. 347-355).

Embryos of avian eggs and mammals are highly sensitive to oxidative stress and hence maintaining a steady reducing environment during the embryonic development is known to confer protection. Although information is completely lacking, proteins of avian egg albumin which have been suggested to play various biological functions, are the major targets for such reducing state during embryogenesis. In this study, we found that ovotransferrin (OTf), the second major protein in egg albumin, undergoes autocleavage at distinct sites upon reduction with thiol-reducing agent or thioredoxin-reducing system. Mass spectral and microsequencing analysis indicated that OTf is able to cleave itself through the unique chemical reactivity of four tripeptides motifs, HTT (residues 209–211), HST (residues 542–544) and two CHT (residues 115–117 and 454–456). Intriguingly, these self-cleavage sites were uniquely located upstream and downstream of the two disulfide kringle domains (residues 115–211 and 454–544) of OTf. These reduction-scissile sequences, His/Cys-X-Thr, are evolutionary conserved self-cleavage motifs found in several autoprocessing proteins including hedgehog proteins. Interestingly, reduction of other two members of transferrin family induced autocleavage patterns, similar to that of OTf, in bovine lactoferrin (bLf) while human lactoferrin (hLf) showed much less self-cleaving activity. This finding is the first to describe that transferrins are a new subset in the class of proteins able to carry out autoprocessing, providing insight into this unusual biochemical process that appears to be a molecular switch involved in triggering a yet unidentified function(s) of OTf as well as bLf.

Keywords: Abbreviations; OTf; ovotransferrin (hen egg white); b-Tf; bovine milk transferrin; h-Tf; human milk transferrin; Trx; thioredoxin; TrxR; thioredoxin reductase; DTT; dithiothreitol; NADPH; nicotinamide adenine dinucleotide phosphate, reduced; SDS-PAGE; sodium dodecylsulfate polyacrylamide gel electrophoresis; MALDI-TOF-MS; Matrix-assisted laser desorption ionization time of flight mass spectrometryOvotransferrin; Transferrin; Autoprocessing; Self-splicing; Redox regulation; Disulfide kringle; Autocleavage motif; Thioredoxin system; Embryo development

Acetohydroxyacid synthase isozyme I from Escherichia coli has unique catalytic and regulatory properties by Valerie Vinogradov; Maria Vyazmensky; Stanislav Engel; Inna Belenky; Alexander Kaplun; Olga Kryukov; Ze'ev Barak; David M. Chipman (pp. 356-363).

AHAS I is an isozyme of acetohydroxyacid synthase which is apparently unique to enterobacteria. It has been known for over 20 years that it has many properties which are quite different from those of the other two enterobacterial AHASs isozymes, as well as from those of “typical�? AHASs which are single enzymes in a given organism. These include a unique mechanism for regulation of expression and the absence of a preference for forming acetohydroxybutyrate. We have cloned the two subunits, ilvB and ilvN, of this Escherichia coli isoenzyme and examined the enzymatic properties of the purified holoenzyme and the enzyme reconstituted from purified subunits. Unlike other AHASs, AHAS I demonstrates cooperative feedback inhibition by valine, and the kinetics fit closely to an exclusive binding model. The formation of acetolactate by AHAS I is readily reversible and acetolactate can act as substrate for alternative AHAS I-catalyzed reactions.

Keywords: Allosteric; Isozyme; Valine inhibition; Oxygenase; Peracetate; R; -PAC

Immunological and physical comparison of monomeric and dimeric phosphagen kinases: Some evolutionary implications by Brianne Wright-Weber; Brenda C. Held; Ashli Brown; Steven H. Grossman (pp. 364-371).

The antigenic and physical properties of several representative invertebrate phosphagen kinases have been examined in order to further characterize the relationship between taxonomic assignment, quaternary protein structure and evolution of this class of enzymes. Antibodies against dimeric arginine kinase from the sea cucumber cross-reacted with dimeric arginine kinase purified from sea urchin eggs, but failed to react with extracts from any species known to contain monomeric arginine kinase. However, strong immunoreactivity was observed when antibodies against purified dimeric arginine kinase were reacted with pure creatine kinase from the human muscle (CK-MM) and brain (CK-BB) as well as extracts from several species known to contain dimeric creatine kinase. Of particular interest with regard to evolution of the phosphagen kinases, we confirm the presence of creatine kinase activity in the very primitive sponge Tethya aurnatium and detect a reaction with antibodies against dimeric, but not monomeric, arginine kinase. This observation is consistent with recent studies of phosphagen kinase evolution. Substrate utilization was very specific with creatine kinase using only creatine. Arginine kinase catalyzed phosphorylation of arginine but enzymes from several species could also phosphorylate canavanine. No activities were detected withd-arginine. Isoelectric points, evaluated for several pure arginine kinases suggest that generally the monomeric forms are more acidic than the dimeric proteins. Heat inactivation of arginine kinase in several species indicated a wide range of stabilities, which did not appear to be correlated with quaternary structure, but rather distinguished by the organism's environment. On the other hand, homodimeric arginine kinase proteins from species inhabiting disparate environments are sufficiently homologous to form a catalytically active hybrid.

Keywords: Abbreviations; AK; ¹; Arginine kinase; ATP; arginine phosphotransferase (EC 2.7.3.3); CK; Creatine Kinase; ATP; creatine phosphotransferase (EC 2.7.3.2)Arginine kinase; Creatine kinase; Cross-reactivity; Physical property

Ring-Toss: Capping highly exposed tyrosyl or tryptophyl residues in proteins with β-cyclodextrin by Zhengping Yi; M.A. Qasim; Sabiha Qasim; T.L. Warrington; Michael Laskowski Jr. (pp. 372-379).

We have used UV difference spectroscopy and fluorescence spectroscopy to study the perturbation by β-cyclodextrin of tyrosyl or tryptophyl residues located at each of the 10 variable consensus contact positions in the third domain of turkey ovomucoid. The goal was to monitor the accessibility of the side chain rings of these residues when located at these positions. The results indicated that the tyrosyl or tryptophyl rings are most highly exposed when located in the P₁ position followed by the P₄ position. It was possible to determine the association constants for β-cyclodextrin binding at these positions. When located at the P₂, P₅, P₆ and P₃′ positions, the rings of the tyrosyl or tryptophyl residues were exposed but less so than at the P₁ or P₄ positions. By contrast, when located at the P₁′, P₂′, P₁₄^′ and P₁₈^′ positions, the tyrosyl or tryptophyl residues were insufficiently exposed to be perturbed by β-cyclodextrin, although they reacted positively to dimethyl sulfoxide solvent perturbation. These findings indicate that β-cyclodextrin perturbation provides a convenient way to detect highly exposed tyrosyls or tryptophyls in proteins. Furthermore, we evaluated the ability of β-cyclodextrin to inhibit the interaction of turkey ovomucoid third domain variants with different P₁ residues. The results showed that the presence of β-cyclodextrin had little effect on the association constant when the P₁ residue was a glycyl residue, but greatly decreased the association constant when the P₁ residue was a tyrosyl or tryptophyl residue. Thus, β-cyclodextrin may be used to selectively modulate the interaction between proteinase inhibitors and their cognate enzymes.

Keywords: Abbreviations; β-CD; β-cyclodextrin; OMTKY3; turkey ovomucoid third domain; DMSO; dimethylsulphoxide; SGPA; Streptomyces griseus; proteinases A; SGPB; Streptomyces griseus; proteinase B; PSA; protein surface water-accessible areas; OMSVP3; silver pheasant ovomucoid third domain; we use; K; _f; to designate the association of β-cyclodextrin with the protein and; K; _a; as the usual association constant for OMTKY3 variants with cognate proteinasesβ-cyclodextrin; Exposed tyrosyl/tryptophyl residues; Proteinase inhibitors; Protein–protein interactions; UV difference spectroscopy; Fluorescence spectroscopy

In situ detection of S-glutathionylated proteins following glutaredoxin-1 catalyzed cysteine derivatization by Niki L. Reynaert; Karina Ckless; Amy S. Guala; Emiel F.M. Wouters; Albert van der Vliet; Yvonne M.W. Janssen-Heininger (pp. 380-387).

S-glutathionylation is rapidly emerging as an important post-translational modification, responsible for transducing oxidant signals. However, few approaches are available that allow visualization of glutathione mixed disulfides in intact cells. We describe here a glutaredoxin1-dependent cysteine derivatization and labeling approach, in order to visualize S-glutathionylation patterns in situ. Using this new method, marked S-glutathionylation was observed in epithelial cells, which was predominant at membrane ruffles. As expected, the labeling intensity was further enhanced in response to bolus oxidant treatments, or in cells overexpressing Nox1 plus its coactivators. In addition, manipulation of endogenous levels of glutaredoxin-1 via RNAi, or overexpression resulted in altered sensitivity to H₂O₂ induced formation of glutathione mixed disulfides. Overall, the derivatization approach described here preferentially detects S-glutathionylation and provides an important means to visualize this post-translational modification in sub-cellular compartments and to investigate its relation to normal physiology as well as pathology.

Keywords: Glutaredoxin; S-glutathionylation; Cysteine derivatization; NADPH oxidase; Oxidant; Cell imaging

Binding modes of V(O) meso-tetrakis( N-methylpyridinium-4-yl)porphyrin to various synthetic DNAs studied by polarized spectroscopy by Tae Gi Park; Jae Hong Ko; Ah Young Ryoo; Jong-Moon Kim; Dae Won Cho; Seog K. Kim (pp. 388-394).

The interactions between water-soluble cationic oxovanadyl[ meso-tetrakis(4- N-methylpyridiumyl)]porphyrin (VOTMPyP) and various synthetic polynucleotide including poly[d(A–T)₂], poly[d(G–C)₂], and poly[d(I–C)₂] were studied using absorption, circular dichroism (CD), and linear dichroism (LD) spectroscopy. When VOTMPyP formed a complex with poly[d(A–T)₂] and poly[d(I–C)₂], a positive CD signal at low [VOTMPyP]/[DNA] ratios ( R ratios) and strong excitonic CD signals at above R≥0.15 were induced. The appearance of the CD spectra of the VOTMPyP-poly[d(G–C)₂] complex were very different: a small negative CD at low R ratios and very small excitonic CD at high R ratios were observed. Considering the facts that the minor grooves of the former two polynucleotides resemble and the major groove of poly[d(I–C)₂] is similar with that of poly[d(G–C)₂], it is conclusive that VOTMPyP binds to the minor groove of all DNA at lower R ratios while they stack at the outside of DNA at higher R ratios. The binding geometry of VOTMPyP to all polynucleotides studied by LD seemed to be homogenous, irrespective of the R ratio. It has been found that VOTMPyP can have five- and six-fluxional coordination states. Comparing the absorption spectra of VOTMPyP complexed with poly[d(A–T)₂] and poly[d(G–C)₂], the distinctive absorptions of the five- and six-coordinated species were observed at lower R ratios which centered at 420–430 nm and 442 nm, respectively. While the six-coordinated VOTMPyP favored the poly[d(A–T)₂], the five-coordinated species favored the poly[d(G–C)₂] at the low R ratios. As the stacked species increased with an increasing R ratio, the six-coordinated species became the major bound species. These observations lead us to conclude that the guanine base′ amino group plays a crucial role not only in determining the binding mode of VOTMPyP but also in the conversion of the six-coordinated species to the five-coordinated species.

Keywords: V(; O)TMPyP; DNA; Linear dichroism; Circular dichroism; Porphyrin; Polynucleotide

Vertebrate freezing survival: Regulation of the multicatalytic proteinase complex and controls on protein degradation by Ashley K. Woods; Kenneth B. Storey (pp. 395-403).

The wood frog , Rana sylvatica, survives weeks of whole body freezing during winter hibernation, expressing numerous metabolic adaptations that deal not only with freezing but with its consequences including organ ischemia and cellular dehydration. The present study analyzes the 20s multicatalytic proteinase (MCP) complex from skeletal muscle to determine how protein degradation is managed in the ischemic frozen state . MCP was partially purified and assayed fluorometrically using three AMC-labeled substrates to compare multiple states: control (5 °C acclimated), 24 h frozen at −2.5 °C, 4 or 8 h thawed at 5 °C, 8 h anoxia, and 40% dehydration. MCP from frozen frogs showed significantly different K_m and V_max values compared with controls; e.g., K_m Z-LLE-AMC increased by 45% during freezing and 52% under anoxia whereas V_max decreased by 40%. After thawing, K_m was restored and V_max rose by 2.2-fold. Incubations promoting protein kinase or phosphatase action on MCP showed that phosphatase treatment strongly increased V_max implicating reversible phosphorylation in MCP regulation during freeze–thaw. Western blotting showed a 36% decrease in MCP protein in muscle from frozen frogs. The 20s MCP preferentially degrades oxidatively-damaged proteins and evidence of impaired function during freezing came from a 1.4-fold increase in protein carbonyl content in muscle and liver during freezing. Ubiquitin and ubiquitin conjugate levels were unchanged in muscle but changed markedly in liver during freeze–thaw.

Keywords: Proteasome; Metabolic suppression; Ubiquitin conjugation; Protein carbonyl; Rana sylvatica; Cryopreservation

Intracellular conformational alterations of mutant SOD1 and the implications for fALS-associated SOD1 mutant induced motor neuron cell death by Fujian Zhang; Haining Zhu (pp. 404-414).

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the selective death of motor neurons. Approximately 10% of ALS cases are familial (fALS) and about 25% of fALS patients inherit autosomal dominant mutations in the gene encoding copper-zinc superoxide dismutase (SOD1). Over 90 different SOD1 mutations have been identified in fALS patients. It has been established that the ALS-linked SOD1 mutations provoke a new toxic function, the nature of which remains unclear. In vitro studies using various biophysical techniques have demonstrated that the SOD1 mutants share a reduced conformational stability. However, conformational alterations of the ALS mutants have not been directly demonstrated in vivo. We employed an SOD1-GFP fusion protein system in this study to monitor the intracellular protein conformation. We demonstrate that the ALS-linked SOD1 mutants adopt different conformations from the wild-type (WT) protein in living cells. Moreover, the conformational alterations of mutant SOD1 render the mutants susceptible to the formation of high-molecular-weight complexes prior to the appearance of detergent-resistant aggregates. Finally, we show that the motor neuron-like cells expressing mutant SOD1 are more susceptible to H₂O₂ induced cell death compared to the cells expressing WT SOD1. This study provides direct evidence of in vivo conformational differences between WT and mutant SOD1. In addition, the SOD1-GFP system can be exploited in future studies to investigate how conformational alterations of mutant SOD1 lead to protein aggregation and to study the potential toxicity of such aggregates in familial ALS.

Keywords: Copper-zinc superoxide dismutase (SOD1); Amyotrophic lateral sclerosis (ALS); Intracellular protein conformation; High-molecular-weight complex; Protein aggregate

An acetylcholinesterase-derived peptide inhibits endocytic membrane activity in a human metastatic breast cancer cell line by Pinar U. Onganer; Mustafa B.A. Djamgoz; Kathryn Whyte; Susan A. Greenfield (pp. 415-420).

Acetylcholinesterase (AChE) is well established as having non-cholinergic functions and is also expressed in breast tumours where its function(s) is not known. Recently, a candidate peptide sequence towards the C-terminal of the AChE molecule has been identified, as the salient site remote from normal catalysis in neurons, and possibly other cells. The main aim of this study was to explore the possibility that ‘AChE-peptide’ might also affect human breast cancer cells. Uptake of the non-cytotoxic tracer horseradish peroxidase (HRP) was used as an index of endocytosis, a key component of the metastatic cascade, representing exocytosis/secretory membrane activity and/or plasma membrane protein turnover. AChE-peptide had no affect on the weakly metastatic MCF-7 human breast cancer cell line. By contrast, application of AChE-peptide to the strongly metastatic MDA-MB-231 cells resulted in a dose-dependent inhibition of HRP uptake; treatment with a scrambled variant of the peptide of comparable amino acid length was ineffective. The action of AChE-peptide was suppressed by lowering the extracellular Ca²⁺ concentration and co-applying a selective antagonist of α7, but not α4/β2, nicotinic receptor. The results suggest that AChE-peptide has a novel, selective bioactivity on breast cancer cells and can potentiate metastatic cell behaviour.

Keywords: Acetylcholinesterase; Breast cancer; HRP; Metastasis; MDA-MB-231; MCF-7

Purification and biochemical characterization of a fibroblast growth factor-binding protein (FGF-BP) from the lactoferrin fraction of bovine milk by Akio Kawakami; Kyoko Hirayama; Fumitaka Kawakami; Hiroshi Kawakami; Michio Fujihara; Kenzo Ohtsuki (pp. 421-431).

By means of gel filtration on a TSK-gel HPLC column in the presence of 8 M urea, a 37-kDa polypeptide (p37) was completely separated from lactoferrin (LF) in the heparin HII fraction of the partially purified LF fraction prepared from bovine milk. Purified p37 was identified as a fibroblast growth factor-binding protein (FGF-BP), since its N-terminal 14 amino acid residues (KKEGRNRRGSKASA) were 100% identical to the corresponding sequence of bovine FGF-BP. It was found, in vitro, that (i) p37 had a higher binding affinity with bFGF than bLF; (ii) p37 functioned as a phosphate acceptor for at least three protein kinases (PKA, CK1 and CK2); (iii) bLF stimulated about 3-fold the PKA-mediated phosphorylation of p37, but suppressed its phosphorylation by CK1; and (iv) galloyl pedunculagin was an effective inhibitor for the phosphorylation of p37 by PKA and CK1. Furthermore, the physiological correlation between p37 and bLF may be regulated through specific phosphorylation of p37 by PKA, since p37 fully phosphorylated by PKA did not bind to bLF in vitro. The sulfatide-induced conformational changes in p37 enabled the phosphorylation of p37 by CK1 and also reduced its ability to bind with bLF in vitro. From these results presented here, it is concluded that (i) p37 (FGF-BP) may be tightly associated with bLF in bovine milk; and (ii) the physiological correlation between p37 and bLF may be regulated by the PKA-mediated full phosphorylation of p37 or by the direct binding of sulfatide to p37 in vivo.

Keywords: Abbreviations; bLF; bovine lactoferrin; CH-3S; cholesterol-3-sulfate; CK1; casein kinase 1; CK2; casein kinase 2; DTT; dithiothreitol; FGF; fibroblast growth factor; FGF-BP; FGF-binding protein; GP; galloyl pedunculagin; PKA; cAMP-dependent protein kinase; PKC; Ca; ²⁺; /phospholipid-dependent protein kinase; p37; 37 kDa polypeptide; QCM; quartz crystal microbalance; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresisBovine lactoferrin; FGF-binding protein; Protein kinase; Phosphorylation; Sulfatide; Bovine milk

Monitoring of cell growth and assessment of cytotoxicity using electrochemical impedance spectroscopy by Manli Guo; Jinhua Chen; Xubin Yun; Kun Chen; Lihua Nie; Shouzhuo Yao (pp. 432-439).

Electrochemical impedance spectroscopy (EIS) was used to monitor the growth of mammalian cancer cells and evaluate the cytotoxicity of chemicals using Fe(CN)₆^3-/4- as a redox probe. Cancer cells, the human hepatocarcinoma cell line (BEL7404), were grown on optically transparent indium tin oxide (ITO) semiconductor slides, which were used as the working electrodes in electrochemical experiments. Attachment and proliferation of cancer cells on ITO surfaces resulted in increase of electron-transfer resistance ( R_et) between the redox probe of Fe(CN)₆^3-/4- in electrolyte solution and ITO electrode surface. For cytotoxicity assessment, cells grown on ITO substrates were further cultured in the presence of different cytotoxicants and electrochemical impedance measurements were carried out at different time intervals. Gemcitabine, a promising antineoplastic drug showing activity against a wide spectrum of human solid tumors, was selected as a model for long-term cytotoxicity effect study, whereas mercury chloride represented a model for acute toxicants. The inhibitions of gemcitabine and mercury chloride on the viability and proliferation of BEL7404 cells were observed from the electrochemical impedance experiments, and the different action modes were discriminated. Additionally, microscope images were also used to observe the effects of these two chemicals on the morphology of the cells. General consistency has been found between the electrochemical impedance response and the morphological observation. Such an impedance method provides a simple and inexpensive way for in vitro assessment of chemical cytotoxicity.

Keywords: Electrochemical impedance; Cytotoxicity; Gemcitabine; Mercury chloride; BEL7404; Indium tin oxide

Determination of H₂O₂-dependent generation of singlet oxygen from human saliva with a novel chemiluminescence probe by Shuna Sun; Xiaohua Li; Guanxin Zhang; Huimin Ma; Deqing Zhang; Zhijuan Bao (pp. 440-444).

Singlet oxygen (¹O₂) has been shown to play an important role in salivary defense system, but its generation process and level from human saliva remain uncertain due to the lack of a reliable detection method. We have previously reported 4,4′(5′)-bis[2-(9-anthryloxy)ethylthio]tetrathiafulvalene (BAET) as a novel chemiluminescence probe for¹O₂. In this work, the probe is successfully used to characterize H₂O₂-dependent generation of¹O₂ from saliva in real time. However, the yield of¹O₂ is found to be very low, for example, being about 0.13 nmol from 200 μL saliva in the presence of 1 mM of hydrogen peroxide over a 5-s reaction period. The result is also compared with that obtained with another¹O₂ probe 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), demonstrating that, besides¹O₂, the other reactive oxygen species such as hydroxyl radical may also be involved in the reaction of saliva with H₂O₂. Furthermore, the present study shows that the selectivity of BAET for¹O₂ is much higher than that of CLA and thus BAET is highly suited for the detection of¹O₂ in the presence of other reactive oxygen species in biological systems.

Keywords: Chemiluminescence; Singlet oxygen generation; Human saliva; 4,4′(5′)-Bis[2-(9-anthryloxy)ethylthio]tetrathiafulvalene; 2-Methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one

“In vitro�? effect of lipid peroxidation metabolites on elongation factor-2 by Sandro Arg�elles; Alberto Machado; Antonio Ayala (pp. 445-452).

Elongation Factor-2 (eEF-2) is the protein that catalyzes the translocation of the ribosome through mRNA. Not all oxidants affect eEF-2, which is extremely sensitive to oxidative stress caused mainly by lipid peroxidant compounds such as cumene hydroperoxide and t-butyl hydroperoxide. Lipid peroxides constitute a potential hazard to living organisms because of their direct reactivity with a variety of biomolecules and the ability to decompose into free radicals and reactive aldehydes. In this “in vitro�? study, we show the effect of three of these aldehydes on the levels of hepatic eEF-2. The results suggest that the toxicity associated with prooxidant-mediated hepatic lipid peroxidation on protein synthesis can originate from the interaction of the aldehydic end products of lipid peroxidation with eEF-2.

Keywords: Abbreviations; eEF-2; Elongation Factor-2; CH; Cumene hydroperoxide; tBHP; t-butyl-hydroperoxide; MDA; malondialdehyde; HNE; 4-hydroxi-noenal; AA; acetaldehyde; DNPH; 2,4-dinitrophenylhydrazine; LP; lipid peroxidesElongation factor-2; Protein synthesis; Lipid peroxides; MDA; Hydroxynonenal; Acetaldehyde; Oxidative stress

Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone Bunodosoma caissarum: Purification and biological characterization by Joacir Stolarz de Oliveira; Andr� Junqueira Zaharenko; Jos� Carlos de Freitas; Katsuhiro Konno; Sonia A. de Andrade; Fernanda C.V. Portaro; Michael Richardson; Osvaldo Augusto Sant'Anna; Denise V. Tambourgi (pp. 453-461).

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes employed gel filtration followed by ion exchange chromatography, being the purity and molecular mass confirmed by SDS-PAGE and mass spectrometry. Protein C1 represented the second major peak of the hemolytic fraction and was previously believed to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular mass of 15495 Da and was assayed for hemolysis, PLA₂ activity and acute toxicity in crabs and mice, showing no activity in these assays. It has an amino terminal with no similarity to all known hemolysins and, therefore, should not be considered a toxin, being its function completely unknown. The protein C3 (19757 Da), that also lacks PLA₂ activity, was recognized by antiserum against Eqt II and presented high hemolytic activity to human erythrocytes (ED₅₀ of 0.270 μg/ml), being named Caissarolysin I (Bcs I). Its activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in presence of erythrocytes pre-treated with the SMase P2, a phospholipase D from the brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins.

Keywords: Hemolysin; Sea anemone; Bunodosoma caissarum; Caissarolysin; Sphingomyelin

N49 phospholipase A₂, a unique subgroup of snake venom group II phospholipase A₂ by Ji-Fu Wei; Xiao-long Wei; Qiu-Yu Chen; Tian Huang; Li-Ya Qiao; Wan-Yu Wang; Yu-Liang Xiong; Shao-Heng He (pp. 462-471).

A novel phospholipase A₂ (PLA₂) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C₁₈ chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A₂s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A₂s are distinct from the other subgroups (D49 PLA₂, S49 PLA₂ and K49 PLA₂) and represent a unique subgroup of snake venom group II PLA₂, named N49 PLA₂ subgroup.

Keywords: Phospholipase A; ₂; Snake venom; Protobothrops mucrosquamatus; Molecular cloning; Phylogenetic analysis; Myotoxicity

Mammalian expression of full-length bovine aggrecan and link protein: Formation of recombinant proteoglycan aggregates and analysis of proteolytic cleavage by ADAMTS-4 and MMP-13 by Hazuki E. Miwa; Thomas A. Gerken; Tru D. Huynh; David M. Flory; Thomas M. Hering (pp. 472-486).

Aggrecan, a large chondroitin sulfate (CS) and keratan sulfate (KS) proteoglycan, has not previously been expressed as a full-length recombinant molecule. To facilitate structure/function analysis, we have characterized recombinant bovine aggrecan (rbAgg) and link protein expressed in COS-7 cells. We demonstrate that C-terminally truncated rbAgg was not secreted. Gel filtration chromatography of rbAgg and isolated glycosaminoglycan (GAG) chains, and their susceptibility to chondroitinase ABC digestion indicate that the GAG chains are predominantly CS, which likely occupy fewer serine residues than native aggrecan. To confirm functionality, we determined that rbAgg bound hyaluronan and recombinant link protein to form proteoglycan aggregates. In addition, cleavage of rbAgg by ADAMTS-4 revealed that the p68 form of ADAMTS-4 preferentially cleaves within the CS-2 domain, whereas the p40 form only effectively cleaves within the interglobular domain (IGD). MMP-13 cleaved rbAgg within the IGD, but cleaved more rapidly at a site within the CS domains, suggesting a role in C-terminal processing of aggrecan. Our results demonstrate that recombinant aggrecan can be used for in vitro analyses of matrix protease-dependent degradation of aggrecan in the IGD and CS domains, and both recombinant aggrecan and link protein can be used to study the assembly of proteoglycan aggregates with hyaluronan.

Keywords: Abbreviations; ADAMTS; a disintegrin and metalloproteinase with thrombospondin motifs; APMA; 4-aminophenylmercuric acetate; BAC; bovine articular chondrocytes; bHA; biotinylated-HA; CAPAGE; composite agarose polyacrylamide gel electrophoresis; CS; chondroitin sulfate; ECM; extracellular matrix; FLAG-rbAgg; FLAG-tagged recombinant aggrecan; G1 domain; N-terminal globular domain of aggrecan; G2 domain; second globular domain of aggrecan; G3 domain; C-terminal globular domain of aggrecan; GAG; glycosaminoglycan; GnHCl; guanidine hydrochloride; HA; hyaluronan; IGD; interglobular domain; KS; keratan sulfate; LP; link protein; MMP; matrix metalloproteinase; OA; osteoarthritis; rbAgg; non-tagged recombinant bovine aggrecanAggrecan; Link protein; Proteoglycan aggregate; Aggrecanase; ADAMTS-4; MMP-13; Aggrecanolysis

Centaureidin promotes dendrite retraction of melanocytes by activating Rho by Yuko Ito; Akiko Kanamaru; Akihiro Tada (pp. 487-494).

Melanosomes synthesized within melanocytes are transferred to keratinocytes through dendrites, resulting in a constant supply of melanin to the epidermis, and this process determines skin pigmentation. During screening for inhibitors of melanosome transfer, we found a novel reagent, centaureidin, that induces significant morphological changes in normal human epidermal melanocytes and inhibits melanocyte dendrite elongation, resulting in a reduction of melanosome transfer in an in vitro melanocyte-keratinocyte co-culture system.Since members of the Rho family of small GTP-binding proteins act as master regulators of dendrite formation, and activated Rho promotes dendrite retraction, we studied the effects of centaureidin on the small GTPases. In in vitro binding assay, centaureidin activated Rho and furthermore, a Rho inhibitor ( C. botulinum C3 exoenzyme), a Rho kinase inhibitor (Y27632) and a small GTPase inhibitor (Toxin B) blocked dendrite retraction induced by centaureidin. These results suggest centaureidin could act via the Rho signaling pathway, and it may directly or indirectly activate Rho.Thus, centaureidin appears to inhibit dendrite outgrowth from melanocytes by activating Rho, resulting in the inhibition of melanosome transfer from melanocytes to keratinocytes.

Keywords: Centaureidin; Dendrite retraction; Cell morphology; Melanosome transfer; Rho

Glutathione transferases with vanadium-binding activity isolated from the vanadium-rich ascidian Ascidia sydneiensis samea by Masafumi Yoshinaga; Tatsuya Ueki; Nobuo Yamaguchi; Kei Kamino; Hitoshi Michibata (pp. 495-503).

Some ascidians accumulate vanadium in vanadocytes, which are vanadium-containing blood cells, at high levels and with high selectivity. However, the mechanism and physiological significance of vanadium accumulation remain unknown. In this study, we isolated novel proteins with a striking homology to glutathione transferases (GSTs), designated AsGST-I and AsGST-II, from the digestive system of the vanadium-accumulating ascidian Ascidia sydneiensis samea, in which the digestive system is thought to be involved in vanadium uptake. Analysis of recombinant AsGST-I confirmed that AsGST-I has GST activity and forms a dimer, as do other GSTs. In addition, AsGST-I was revealed to have vanadium-binding activity, which has never been reported for GSTs isolated from other organisms. AsGST-I bound about 16 vanadium atoms as either V(IV) or V(V) per dimer, and the apparent dissociation constants for V(IV) and V(V) were 1.8×10⁻⁴ M and 1.2×10⁻⁴ M, respectively. Western blot analysis revealed that AsGSTs were expressed in the digestive system at exceptionally high levels, although they were localized in almost all organs and tissues examined. Considering these results, we postulate that AsGSTs play important roles in vanadium accumulation in the ascidian digestive system.

Keywords: Ascidian; Vanadium; Metal accumulation; Digestive system; Glutathione transferase

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae by Hana Flegelova; Rosine Haguenauer-Tsapis; Hana Sychrova (pp. 504-516).

Na⁺/H⁺ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1–3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.

Keywords: NHE exchangers; Heterologous expression; Protein trafficking; npi1; mutation; Saccharomyces cerevisiae

Glycosaminoglycan-binding properties and aggrecanase activities of truncated ADAMTSs: Comparative analyses with ADAMTS-5, -9, -16 and -18 by Weilan Zeng; Chris Corcoran; Lisa A. Collins-Racie; Edward R. LaVallie; Elisabeth A. Morris; Carl R. Flannery (pp. 517-524).

Aggrecanases are ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motifs) proteases capable of primary (patho)physiological cleavage at specific Glu- Xaa bonds within the core protein of the hyaluronan-binding proteoglycan aggrecan. Accumulating evidence suggests that regulation of the activity of one such aggrecanase, ADAMTS-4 (or Aggrecanase-1), involves post-translational C-terminal processing (truncation) which modulates both glycosaminoglycan (GAG)-binding affinity and enzymatic activity. In the present study, we compared the effects of C-terminal truncation on the GAG-binding properties and aggrecanase activity of ADAMTS-5 (Aggrecanase-2) relative to three other ADAMTS family members, ADAMTS-9, ADAMTS-16 and ADAMTS-18. Full-length recombinant human ADAMTS-5 ( M_r ∼85 kDa; ADAMTS-5(p85)) underwent autolytic cleavage during expression by CHO/A2 cells, and co-purified with C-terminally truncated (tr) isoforms of M_r ∼60 kDa (ADAMTS-5(p60) and M_r ∼45 kDa (ADAMTS-5(p45)). All three ADAMTS-5 isoforms bound to sulfated GAGs (heparin and chondroitin sulfate (CS)). An ADAMTS-5(p45) structural mimetic, terminating at Phe⁶²⁸ and comprising the catalytic domain, disintegrin-like domain and thrombospondin type I repeat (TSR)-1 domain (designated trADAMTS-5F⁶²⁸), also bound to heparin, and exhibited potent aggrecanase activity toward cleavage sites both in the aggrecan CS-2-attachment region (at Glu¹⁷⁷¹–Ala¹⁷⁷²) and in the interglobular domain (at Glu³⁷³–Ala³⁷⁴). Further truncation (deletion of the TSR-1 domain) of ADAMTS-5 significantly reduced aggrecanase activity, although appreciable GAG (heparin)-binding affinity was maintained. Other TSR-1 domain-bearing truncated ADAMTS constructs demonstrating either positive GAG-binding ability (trADAMTS-9^F649) or negligible GAG-affinity (trADAMTS-16^F647 and trADAMTS-18^F650) displayed comparably low aggrecanase activities. Thus, the presence of TSR-1 on truncated ADAMTSs appears to be necessary, but not sufficient, for effective aggrecanase-mediated catalysis of target Glu- Xaa bonds. Similarly, GAG-binding ability, irrespective of the presence of a TSR-1 domain, does not necessarily empower truncated ADAMTSs with proficient aggrecanase activity.

Keywords: ADAMTS; Aggrecan; aggrecanase; Glycosaminoglycan-binding; Metalloproteinase; Proteoglycan

Sections

Personal tools

BBA - General Subjects (v.1760, #3)

Sections

Personal tools

Local resources

BBA - General Subjects (v.1760, #3)