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BBA - General Subjects (v.1770, #4)

Editorial Board (pp. ii).

Low molecular weight chitosans—Preparation with the aid of pronase, characterization and their bactericidal activity towards Bacillus cereus and Escherichia coli by Acharya B. Vishu Kumar; Mandyam C. Varadaraj; Lalitha R. Gowda; Rudrapatnam N. Tharanathan (pp. 495-505).

The homogeneous low molecular weight chitosans (LMWC) of molecular weight 9.5–8.5 kDa, obtained by pronase catalyzed non-specific depolymerization (at pH 3.5, 37 °C) of chitosan showed lyses of Bacillus cereus and Escherichia coli more efficiently (100%) than native chitosan (<50%). IR and¹H-NMR data showed decrease in the degree of acetylation (14–19%) in LMWC compared to native chitosan (∼26%). Minimum inhibitory concentration of LMWC towards 10⁶ CFU ml−¹ of B. cereus was 0.01% (w/v) compared to 0.03% for 10⁴ CFU ml−¹ of E. coli. SEM revealed pore formation as well as permeabilization of the bacterial cells, as also evidenced by increased carbohydrate and protein contents as well as the cytoplasmic enzymes in the cell-free supernatants. N-terminal sequence analyses of the released proteins revealed them to be cytoplasmic/membrane proteins. Upon GLC, the supernatant showed characteristic fatty acid profiles in E. coli, thus subscribing to detachment of lipopolysaccharides into the medium, whereas that of B. cereus indicated release of surface lipids. The mechanism for the observed bactericidal activity of LMWC towards both Gram-positive and Gram-negative bacteria has been discussed.

Keywords: Abbreviations; M; _r; Molecular weight; DA; Degree of acetylation; DP; Degree of polymerization; CFU; Colony forming units; MIC; Minimum inhibitory concentration; HPSEC; High performance size exclusion chromatography; SEM; Scanning electron microscopy; GPC; Gel permeation chromatography; GLC; Gas–liquid chromatography; LPS; lipopolysaccharidesLow molecular weight chitosan; Pronase; Structure; Bactericidal activity; Mechanism

Giant extracellular Glossoscolex paulistus Hemoglobin (HbGp) upon interaction with cethyltrimethylammonium chloride (CTAC) and sodium dodecyl sulphate (SDS) surfactants: Dissociation of oligomeric structure and autoxidation by Patricia S. Santiago; Leonardo M. Moreira; Erika V. de Almeida; Marcel Tabak (pp. 506-517).

The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein–surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein–surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein–surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein–surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the anionic one. This is opposite as observed for several other hemoproteins. The surfactants at low concentrations produce the oligomeric dissociation, which facilitates the iron oxidation, an important factor modulating further oligomeric protein dissociation.

Keywords: Extracellular hemoglobin; Ionic surfactants; Oligomeric dissociation; Heme autoxidation; Optical spectroscopies; Heme coordination

Focal adhesion kinase determines the fate of death or survival of cells in response to TNFα in the presence of actinomycin D by Reiko Takahashi; Yoshiko Sonoda; Daiju Ichikawa; Naomi Yoshida; Aizu-Yokota Eriko; Kasahara Tadashi (pp. 518-526).

We speculated that focal adhesion kinase (FAK) might play a critical role in the TNFα-induced cell death. In this study, we found that FAK−/− cells are more sensitive to TNFα-induced apoptosis in the presence of actinomycin D (Act D) compared to FAK+/− cells. Prosurvival pathways are activated by the rapid recruitment of complex I, comprising TNFR1, TRADD, RIP and TRAF2, which leads to the activation of the NF-κB pathway. On the other hand, proapoptotic pathways are activated by complex II, the death-inducing signaling complex (DISC), which contains TNFR1, TRADD, RIP, and FADD, and procaspase-8 proteins. As TNFR1, TRADD, and RIP are included in both Complex I and DISC, we speculated that RIP might be a key protein. Coimmunoprecipitation assays revealed that RIP is included in complex I in FAK+/− cells, and FAK was associated with RIP. On the other hand, RIP is included in DISC in FAK−/− cells. FAK might be a key protein in the formation of complex I and the activation of NF-κB. Furthermore, Akt was activated in FAK+/− cells, but not FAK−/− cells. In conclusion, we first demonstrated that FAK determines the pathway leading to death or survival in TNFα/ActD-stimulated fibroblasts.

Keywords: FAK; TNFα; NF-κB; RIP; Apoptosis; DISC

Identification and biochemical analysis of a mitochondrial endonuclease of Podospora anserina related to curved-DNA binding proteins by Patricia Laquel-Robert; Carole H. Sellem; Annie Sainsard-Chanet; Michel Castroviejo (pp. 527-542).

We purified and characterized previously from Podospora anserina mitochondria an endonuclease, active on single-stranded, double-stranded and flap DNA, with RNAse H activity, named P49 according to the major 49 kDa band observed on SDS-PAGE. Edman sequencing allowed us to identify the corresponding gene called nuc49. Here we report the properties of the (His)-tagged NUC49 protein expressed in E. coli. We show that this protein does exhibit an endonuclease activity on plasmid DNA, circular recessed and flap M13 substrate with short protruding single strand. However, in contrast to the mt endonuclease purified fraction it does not present RNase H activity and does not cleave linear flap substrate. The activity differences between the protein expressed in E. coli and the mitochondrial endonuclease fraction previously described are discussed. NUC49 presents a strong homology with the S. pombe CDB4 curved DNA binding protein which belongs to a large family including the human cell cycle protein PA2G4 and is able to bind curved DNA. The results constitute the first description of a mitochondrial endonuclease activity associated to this family of proliferation associated homologous proteins. The function of this endonuclease either in recombination, repair or mt DNA rearrangements remains to be determined.

Keywords: Endonuclease; DNA binding protein; Mitochondria; Fungus

Inositol-1 (or 4)-monophosphatase from Glycine max embryo axes is a phosphatase with broad substrate specificity that includes phytate dephosphorylation by Ignacio Islas-Flores; Marco A. Villanueva (pp. 543-550).

A phosphate-hydrolyzing activity from Glycine max embryo axes was purified by a series of chromatographic steps and electroelution from activity gels, and demonstrated to be an inositol-1 (or 4)-monophosphatase by partial internal amino acid sequence. This enzyme hydrolyzed ATP, sodium pyrophosphate (NaPPi), inositol hexakisphosphate, and inositol 1-monophosphate, but not p-nitrophenyl phosphate, ADP, AMP or glucose 6-P. Using NaPPi as substrate, the highly purified protein hydrolyzed up to 0.4 mmol phosphate min−¹ mg−¹ protein and had a Km_avg of 235 μM for NaPPi. Since NaPPi is relatively inexpensive and readily available, we used this as substrate for the subsequent characterization. We observed the following: (a) specific inhibition by Li and NaF but not by butanedione monoxime, or orthovanadate; (b) activation by Cu²⁺ and Mg²⁺; (c) optimum activity at pH 7.4; and (d) temperature stability after 1-h incubations at 37–80 °C, with maximum activity at 37 °C. The partially purified protein was detected by in-gel activity assays and the band was electroeluted to yield a highly purified protein. Analysis by SDS-PAGE and native IEF-PAGE yielded a single major polypeptide of 29 kDa and pI ∼5.9, respectively. In addition, in-gel activity from embryo axes and whole hypocotyls at early germination times revealed one high and one intermediate molecular weight isoform, but only the intermediate one corresponded to IMPase. Throughout the post-imbibition period, the activity of the high molecular weight isoform disappeared and IMPase increased, indicating an increasing expression of the enzyme as germination and growth proceeded. These data indicate that the inositol-1 (or 4)-monophosphatase present in the embryo axis of G. max has a wide phosphate substrate specificity, and may play an important role in phosphate metabolism during the germination process.

Keywords: Abbreviations; ATP; adenosine 5′-triphosphate; ADP; Adenosine 5′-(trihydrogen diphosphate); AMP; adenosine monophosphate; DEAE; diethylaminoethyl; DTT; dithiothreitol; EDTA; ethylenediamine-; N,N,N′,N′; -tetraacetic acid; EGTA; ethyleneglycol-bis(β-aminoethyl)-; N,N,N′,N′; -tetracetic acid; IEF; isoelectric focusing; IMP; D; -; myo; -inositol 1-monophosphate; IMPase; inositol-1 (or 4)-monophosphatase; glucose 6-P; D; -glucose 6-phosphate; β-ME; β-mercaptoethanol; MSB; microtubule-stabilizing buffer; NaPPi; sodium pyrophosphate; PAGE; polyacrylamide gel electrophoresis; Pi; phosphate; PIPES; piperazine-1,4-bis(2-ethanesulfonic acid); p; -NPP; p; -nitrophenyl phosphate; PPase; pyrophosphatase; SDS; sodium dodecyl sulfateGermination; Glycine max; IMPase; Seeds; Phosphatase; Phytate

Binding properties of Clostridium botulinum type C progenitor toxin to mucins by Toshio Nakamura; Noriko Takada; Takashi Tonozuka; Yoshiyuki Sakano; Keiji Oguma; Atsushi Nishikawa (pp. 551-555).

It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327–333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N-acetylneuraminic acid, N-glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl α2–3 lactose and sialyl α2–6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures.

Keywords: Abbreviations; HA; hemagglutinin; NTNH; nontoxic nonhemagglutinin; NT; neurotoxin; BSM; bovine submaxillary mucin; PGM; porcine gastric mucin Clostridium botulinum; Hemagglutinin; O; -linked oligosaccharide; Mucin; BSM; PGM

Thymoquinone attenuates proinflammatory responses in lipopolysaccharide-activated mast cells by modulating NF-kappaB nuclear transactivation by Mohamed A. El Gazzar; Rabab El Mezayen; Mark R. Nicolls; Stephen C. Dreskin (pp. 556-564).

Activated mast cells play an important role in the development and maintenance of chronic inflammation by releasing proinflammatory cytokines such as Tumor necrosis factor alpha (TNFα). TNFα is a key mediator of immune and inflammatory responses as it controls the expression of inflammatory genes network and its overproduction contributes significantly to the pathological complications observed in many inflammatory diseases. We have previously shown that thymoquinone (TQ), which has broad anti-inflammatory activities, attenuates allergic inflammation in mice. In the present study, we investigated the effect of TQ on LPS-induced TNFα production in the rat basophil cell line, RBL-2H3. Stimulation of RBL-2H3 cells with LPS markedly increased TNFα production. TQ treatment significantly inhibited LPS-induced TNFα mRNA expression and protein production. To understand the mechanism by which TQ inhibited TNFα production, we examined its effects on activation of NF-κB transcription factor, which has been shown to be involved in regulating TNFα responses. LPS activated the NF-κB pathway, resulting in accumulation of NF-κB p65 and p50 subunits in the nucleus and activation of TNFα promoter. TQ administration to LPS-stimulated cells did not noticeably alter NF-κB cytosolic activation or nuclear expression as demonstrated by western blot analysis. Instead, TQ significantly increased the amount of the repressive NF-κB p50 homodimer, and simultaneously decreased the amount of transactivating NF-κB p65:p50 heterodimer, bound to the TNFα promoter as revealed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Transient transfection of RBL-2H3 cells with TNFα promoter-driven luciferase gene constructs demonstrated that one of the three NF-κB binding sites in the TNFα promoter, the κB3 site, played a major role in the induction of TNFα promoter-driven luciferase gene expression by LPS, as well as in mediating the inhibitory effects of TQ on TNFα production, as TQ had minimal effect on the TNFα promoter-luciferase construct that lacks the κB3 site. Together, these results suggest that TQ attenuates the proinflammatory response in LPS-stimulated mast cells by modulating nuclear transactivation of NF-κB and TNFα production.

Keywords: Inflammation; Thymoquinone; Mast cell; TNFα; NF-kappaB; Transcriptional regulation

The vicinal hydroxyl group is prerequisite for metal activation of Clostridium thermocellum acetylxylan esterase by Peter Biely; Mária Mastihubová; Vladimír Puchart (pp. 565-570).

Positional specificity of NodB-like domain of a multidomain xylanase U from Clostridium thermocellum ( CtAxe) was investigated. Of three monoacetates of 4-nitrophenyl β-d-xylopyranoside the acetylxylan esterase domain showed a clear preference for the 2-acetate. Moreover, the enzyme was significantly activated by Co²⁺. Acetylated methyl β-d-xylopyranosides were deacetylated slightly better at position 3 than at position 2, suggesting that the enzyme binds the substrate with the small methyl aglycone also in the opposite orientation. Nevertheless, both positions 2 and 3 of methyl β-d-xylopyranoside were deacetylated much faster in the presence of the activating metal ion. In contrast, replacement of the hydroxyl group at either of these positions with fluorine or hydrogen, as well as acetylation of both positions, abolished the enzyme activity, regardless the absence or the presence of Co²⁺. Thus, the presence of the free vicinal hydroxyl group seems to be a prerequisite not only for an efficient deacetylation of position 2 or 3, but also for the activation of the enzyme with cobalt ion. The demonstrated involvement of the vicinal hydroxyl groups in the mechanism of deacetylation is in accord with 3-D structures of CtAxe as well as other CE4 metal-dependent deacetylases.

Keywords: Abbreviations; AcXE; acetylxylan esterase; CE4; carbohydrate esterase family 4; Ct; Axe; CE4 catalytic module of bifunctional; Clostridium thermocellum; F1/YS xylanase U; Sl; AxeA; Streptomyces lividans; 1326 acetylxylan esterase A Clostridium thermocellum; Streptomyces lividans; Acetylxylan esterase; Carbohydrate esterase family 4; Positional specificity

Vitamin B₆ suppresses apoptosis of NM-1 bovine endothelial cells induced by homocysteine and copper by Naoko Endo; Kazuo Nishiyama; Masaaki Okabe; Mitsuharu Matsumoto; Hiroaki Kanouchi; Tatsuzo Oka (pp. 571-577).

Hyperhomocysteinemia is an important risk factor for atherosclerosis. We previously reported that formation of early atherosclerosis in the rat aorta was associated with hyperhomocysteinemia and reduction of antioxidant activity caused by low concentration of vitamin B₆ in vivo. In the present study, we examined effects of vitamin B₆ on apoptosis of bovine endothelial cells (NM-1 cells) treated with homocysteine and copper. Homocysteine and copper induced extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. Cell viability was reduced to 30% compared to that of control cells. On the other hand, pyridoxal treatment as well as EDTA treatment increased viability of NM-1 cells treated with homocysteine and copper to about 60%, and significantly decreased extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. The treatment of catalase recovered cell viability and reduced the level of extracellular hydrogen peroxide and intracellular ROS. Cell death by homocysteine and copper was confirmed to be due to apoptosis by evaluation of DNA fragmentation and by TUNEL assay. However, apoptosis of NM-1 cells induced by homocysteine and copper was due to a caspase-independent pathway as it was not inhibited by the caspase inhibitor, Z-VAD-fmk. Apoptosis of NM-1 cells induced by homocysteine and copper accompanied with mitochondrial permeability but not cytochrome c release. These results suggest that pyridoxal treatment suppresses apoptosis of NM-1 cells induced by homocysteine and copper, most likely through antioxidant effects.

Keywords: Vitamin B; ₆; Homocysteine; Copper; Antioxidant; Apoptosis; NM-1 cells

Protective effects of carotenoids from saffron on neuronal injury in vitro and in vivo by Takashi Ochiai; Hiroshi Shimeno; Ken-ichi Mishima; Katsunori Iwasaki; Michihiro Fujiwara; Hiroyuki Tanaka; Yukihiro Shoyama; Akihisa Toda; Reiko Eyanagi; Shinji Soeda (pp. 578-584).

Crocus sativus L. (saffron) has been used as a spice for flavoring and coloring food preparations, and in Chinese traditional medicine as an anodyne or tranquilizer. Our previous study demonstrated that crocin, a carotenoid pigment of saffron, can suppress the serum deprivation-induced death of PC12 cells by increasing glutathione (GSH) synthesis and thus inhibiting neutral sphingomyelinase (nSMase) activity and ceramide formation. The carotenoid pigments of saffron consist of crocetin di-(β-d-glucosyl)-ester [dicrocin], crocetin-(β-d-gentiobiosyl)-(β-d-glucosyl)-ester [tricrocin] and crocetin-di-(β-d-gentiobiosyl)-ester [crocin]. Saffron also contains picrocrocin, the substance causing saffron's bitter taste. In this study, to confirm whether neuroprotective effects of saffron are caused solely by crocin, we examined the antioxidant and GSH-synthetic activities of these crocins in PC12 cells under serum-free and hypoxic conditions. Measurements of cell viability, peroxidized membrane lipids and caspase-3 activity showed that the rank order of the neuroprotective potency at a concentration of 10 μM was crocin>tricrocin>dicrocin and picrocrocin (the latter two crocins had a little or no potency). In addition, we show that among these saffron's constituents, crocin most effectively promotes mRNA expression of γ-glutamylcysteinyl synthase (γ-GCS), which contributes to GSH synthesis as the rate-limiting enzyme, and that the carotenoid can significantly reduce infarcted areas caused by occlusion of the middle cerebral artery (MCA) in mice.

Keywords: Saffron; Crocetin glycosides; Hypoxia; Neuronal cell death; Glutathione; Cerebral ischemia

Structural and functional properties of BaTX, a new Lys49 phospholipase A₂ homologue isolated from the venom of the snake Bothrops alternatus by Luis Alberto Ponce-Soto; Bruno Lomonte; José María Gutiérrez; Lea Rodrigues-Simioni; José Camillo Novello; Sergio Marangoni (pp. 585-593).

BaTX PLA₂, a K49 phospholipase A₂ homologue was purified from Bothrops alternatus venom after two chromatographic steps, molecular exclusion on Superdex 75 and reverse phase HPLC on μ-Bondapack C-18. A molecular mass of 13898.71 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that BaTX has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA₂. The complete amino acid sequence of BaTX PLA₂ contains 121 residues, resulting in a calculated pI value of 8.63. This sequence shows high identity values when compared to other K49 PLA₂s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA₂s. The sequence was SLFELGKMIL QETGKNPAKS YGAYYCYCGW GGQGQPKDAT DRCCYVHKCC YKKLTGCNPK KDRYSYSWKD KTIVCGENNS CLKELCECDK AVAICLRENL NTYNKKYRYY LKPLCKKADA C. In mice, BaTX induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. The LD₅₀ of BaTX was 7 μg/g body weight, by intravenous route. In vitro, the toxin caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparations. The blockage 50% was achieved at a concentration of 0.03 μM: 40±0.4 min and 0.07 μM: 35±0.3 min. Moreover, this protein induced a rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. Thus, the combined structural and functional information obtained identify BaTX as a new member of the K49 PLA₂ family, which presents the typical bioactivities described for such proteins.

Keywords: Phospholipase A; ₂; Lys-49; Neurotoxin; Myotoxin; Snake venom; Bothrops alternatus

Glutamine, insulin and glucocorticoids regulate glutamine synthetase expression in C2C12 myotubes, Hep G2 hepatoma cells and 3T3 L1 adipocytes by Yanxin Wang; Malcolm Watford (pp. 594-600).

The cell-specific regulation of glutamine synthetase expression was studied in three cell lines. In C2C12 myotubes, glucocorticoids increased the abundance of both glutamine synthetase protein and mRNA. Culture in the absence of glutamine also resulted in very high glutamine synthetase protein abundance but mRNA levels were unchanged. Glucocorticoids also increased the abundance of glutamine synthetase mRNA in Hep G2 hepatoma cells but this was not reflected in changes in protein abundance. Culture of Hep G2 cells without glutamine resulted in very high levels of protein, again with no change in mRNA abundance. Insulin was without effect in both C2C12 and Hep G2 cells. In 3T3 L1 adipocytes glucocorticoids increased the abundance of both glutamine synthetase mRNA and protein, insulin added alone had no effect but in the presence of glucocorticoids resulted in lower mRNA levels than seen with glucocorticoids alone, although protein levels remained high under such conditions. In contrast to the other cell lines glutamine synthetase protein levels were relatively unchanged by culture in the absence of glutamine. The results support the hypothesis that in myocytes, and hepatomas, but not in adipocytes, glutamine acts to moderate glutamine synthetase induction by glucocorticoids.

Keywords: Glutamine; Glutamine synthetase; Skeletal muscle; Liver; Adipocyte; Insulin; Glucocorticoid

Biophysical characterization of MDR breast cancer cell lines reveals the cytoplasm is critical in determining drug sensitivity by Helen M. Coley; Fatima H. Labeed; Hilary Thomas; Michael P. Hughes (pp. 601-608).

Dielectrophoresis (DEP) was used to examine a panel of MCF-7 cell lines comprising parental MCF-7 cells and MDR derivatives: MCF-7TaxR (paclitaxel-resistant, P-glycoprotein (P-gp) positive), MCF-7DoxR (doxorubicin-resistant MRP2 positive) plus MCF-7 MDR1 ( MDR1 transfected, P-gp positive). MCF-7DoxR and MCF-7 MDR1 were broadly cross-resistant to natural product anticancer agents, whereas MCF-7TaxR cells were not, contrary to P-gp expression. Whilst DEP revealed modest membrane changes in MDR sub-lines, we saw significant changes in their cytoplasmic conductivity: MCF-7TaxRMDR1 showed an increase. Thus, altered membrane potential is associated with an MDR phenotype, but in a complex manner. DEP data suggest a model whereby relative increases in cytoplasmic conductivity are correlated with MDR, whilst relative decreases equate with a sensitised phenotype e.g. MCF-7TaxR. Moreover, extent of anthracycline accumulation was inversely related to cytoplasmic conductivity. These data are representative of a model where drug sensitivity is associated with low ionic conductance (reduced cellular trafficking and ion transport) and substantial anthracycline accumulation. For classical MDR i.e. MCF-7 MDR1, we saw the reverse picture. Thus, the drug resistance phenotypes of this panel of MCF-7 lines can be delineated by assessment of cytoplasmic biophysical properties using DEP.

Keywords: Dielectrophoresis; P-gp; MDR; Drug sensitivity

The critical period for thyroid hormone responsiveness through thyroid hormone receptor isoform α in the postnatal small intestine by Kazuki Mochizuki; Eriko Yagi; Naomi Sakaguchi; Hiroko Mochizuki; Satsuki Takabe; Sachi Kuranuki; Takuji Suzuki; Masaya Shimada; Toshinao Goda (pp. 609-616).

During second and third weeks after birth in rats, serum thyroid hormone level is elevated. In this study, we investigated the jejunal expression of thyroid hormone receptor (TR) α in developing rats. The TRα-1 mRNA level and TRα-1/TRα-2 mRNA ratio increased two-fold from 5 to 13 days after birth. This high level of TRα-1 mRNA was maintained until 20 days and then decreased to the basal level by the end of weaning period at 27 days; however, the level of TRα-2 mRNA remained unchanged throughout the developmental period. The increase in the TRα-1/TRα-2 mRNA ratio from 5 to 13 days was accompanied by an initial rise in the levels of mRNA for hexose transporters in the jejunum. Administration of T₃ during the suckling period (8–13 days) caused a 50% increase in the TRα-1/TRα-2 mRNA ratio, while administration of T₃ on days 12–17 and days 16–21, but not on days 22–27, caused a two to four-fold increase in the levels of mRNA for hexose transporters. These results suggest that a transient variation in the TRα-1/TRα-2 expression ratio is closely related to the critical period of thyroid hormone responsiveness for hexose transporters expression in the developing rat jejunum.

Keywords: Thyroid hormone; TRα; Postnatal development; Hexose transporter; Small intestine

Structural characterization of a rhamnose-binding glycoprotein (lectin) from Spanish mackerel ( Scomberomorous niphonius) eggs by Takatomo Terada; Yasuharu Watanabe; Hiroaki Tateno; Takako Naganuma; Tomohisa Ogawa; Koji Muramoto; Hisao Kamiya (pp. 617-629).

A rhamnose-binding glycoprotein (lectin), named SML, was isolated from the eggs of Spanish mackerel ( Scomberomorous niphonius) by affinity and ion-exchange chromatographies. SML was composed of a non-covalently linked homodimer. The SML subunit was composed of 201 amino acid residues with two tandemly repeated domains, and contained 8 half-Cys residues in each domain, which is highly homologous to the N-terminal lectin domain of calcium-independent α-latrotoxin receptor in mammalian brains. Each domain has the same disulfide bonding pattern; Cys10–Cys40, Cys20–Cys99, Cys54–Cys86 and Cys67–Cys73 were located in the N-terminal domain, and Cys108–Cys138, Cys117–Cys195, Cys152–Cys182 and Cys163–Cys169 were in the C-terminal domain. SML was N-glycosylated at Asn168 in the C-terminal domain. The structure of the sugar chain was determined to be NeuAc-Galβ1-4GlcNAcβ1-2Manα1-6-(NeuAc-Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc-Asn.

Keywords: Abbreviations; BCA; bincinchonic acid; BSA; bovine serum albumin; CAM; S; -carboxamidomethylated; CRD; carbohydrate-recognition domain; CSL; chum salmon egg lectin; DHBA; 2,5-dihydroxybenzoic acid; DMB; 1,2-diamino-4,5-methylene dioxybenzene; 2D; two dimensional; kDa; kilodalton; MALDI-TOF; matrix-assisted laser desorption ionization time of flight; 2-ME; 2-mercaptoethanol; NeuAc; N; -acetylneuraminic acid; NeuGc; N; -glycolylneuraminic acid; PA; pyridyl amine; PE; pyridyl ethyl; RBL; rhamnose-binding lectin; RCLD; RBL CRD-like domain; RP-HPLC; reversed-phase high-performance liquid chromatography; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; SML; Spanish mackerel egg lectin; STL; steelhead tout egg lectin; SUEL; sea urchin egg lectin; TBS; 20 mM Tris–HCl buffer (pH 8.0) containing 0.5 M NaCl; TFA; trifluoroacetic acid; WCL; white-spotted charr egg lectinAnimal lectin; Rhamnose-binding lectin; Spanish mackerel; Glycoprotein; Disulfide bonds; N; -linked sugar chains

An exodeoxyribonuclease from Streptomyces coelicolor: Expression, purification and biochemical characterization by Zuzana Brnáková; Andrej Godány; Jozef Timko (pp. 630-637).

Streptomyces coelicolor A3(2) produces several intra and extracellular enzymes with deoxyribonuclease activities. The examined N-terminal amino acid sequence of one of extracellular DNAases (TVTSVNVNGLL) and database search on S. coelicolor genome showed a significant homology to the putative secreted exodeoxyribonuclease. The corresponding gene ( exoSc) was amplified, cloned, expressed in Escherichia coli, purified to homogeneity and characterized. Exonuclease recExoSc degraded chromosomal, linear dsDNA with 3′-overhang ends, linear ssDNA and did not digest linear dsDNA with blunt ends, supercoiled plasmid ds nor ssDNA. The substrate specificity of recExoSc was in the order of dsDNA>ssDNA>3′-dAMP. The purified recExoSc was not a metalloprotein and exhibited neither phosphodiesterase nor RNase activity. It acted as 3′-phosphomonoesterase only at 3′-dAMP as a substrate. The optimal temperature for its activity was 57 °C in Tris–HCl buffer at optimal pH=7.5 for either ssDNA or dsDNA substrates. It required a divalent cation (Mg²⁺, Co²⁺, Ca²⁺) and its activity was strongly inhibited in the presence of Zn²⁺, Hg²⁺, chelating agents or iodoacetate.

Keywords: Streptomyces coelicolor; nuclease; recExoSc; Exodeoxyribonuclease; Purification

Alterations in proteoglycan synthesis selectively impair FSH-induced particulate cAMP-phosphodiesterase 4 (PDE4) activation in immature rat Sertoli cells by Guénaëlle Levallet; Jérôme Levallet; Pierre-Jacques Bonnamy (pp. 638-648).

FSH-induced upregulation of cAMP-PDE4 activities was decreased in cultured Sertoli cells when alteration of cell proteoglycans (PGs) metabolism was simultaneously induced either by para-nitrophenyl β-d-xyloside (PNPX) or by sodium chlorate. This effect was restricted to the particulate PDE4 activities and its timing was consistent with the half-life of Sertoli cell PGs. It did not result from alterations in Pde4d variants expression, the major FSH-regulated PDE4 in Sertoli cells. Moreover, lack of changes in the particulate levels of major immunoreactive 75 kDa and 90 kDa PDE4D proteins, corresponding likely to short PDE4D1 and long PDE4D3/D8/D9 isoforms respectively, suggested that the decrease in FSH-stimulated of PDE4 activities in chlorate- and PNPX-treated cells at the end of the 24-h incubation period resulted from the increased reversal of the activated particulate PDE4(D) activities back to unstimulated levels. By controlling FSH-stimulated particulate PDE4 inactivation through a still unknown mechanism (sustained activation of PKA or reduction of phosphoprotein phosphatase activities), cell PGs could be involved in the alteration of cAMP response to FSH accompanying the transition of Sertoli cells from proliferative to non-proliferative differentiated state.

Keywords: Abbreviations; PDE4; type 4 cAMP-dependent phosphodiesterases; HSPG; heparan sulfate proteoglycan; PNPX; para; -nitrophenyl-β-; d; -xylosidephosphodiesterase 4D (PDE4D); proteoglycan (PG); Sertoli cell; FSH; rat

Differential expression of NF-κB in mycobacteria infected THP-1 affects apoptosis by Rohan Dhiman; Manoj Raje; Sekhar Majumdar (pp. 649-658).

The present study was conducted to see the role of NF-κB in virulent ( Mycobacterium tuberculosis H37Rv) and avirulent ( M. tuberculosis H37Ra) mycobacterial infection in THP-1 cells. To inactivate NF-κB, pCMV-IκBαM dn containing THP-1 cell line was generated which showed marked increase in apoptosis with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Infected THP-1-IκBαM dn cells showed decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and enhanced TNF-α production. Increase in apoptosis of infected THP-1-IκBαM dn cells resulted in inhibition of intracellular mycobacterial growth. Differential NF-κB activation potential was observed with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Both the strains activated NF-κB after 4 h in THP-1 cells however after 48 h only M. tuberculosis H37Rv activated NF-κB which lead to up-regulation of bcl-2 family anti-apoptotic member, bfl-1/A1. Our results indicated that NF-κB activation may be a determinant factor for the success of virulent mycobacteria within macrophages.

Keywords: THP-1 cells; Mycobacteria; NF-κB; Apoptosis; bfl-1/A1

Lycopene as a natural protector against γ-radiation induced DNA damage, lipid peroxidation and antioxidant status in primary culture of isolated rat hepatocytes in vitro by M. Srinivasan; A. Ram Sudheer; K. Raveendran Pillai; P. Raghu Kumar; P.R. Sudhakaran; V.P. Menon (pp. 659-665).

The present study was designed to evaluate the radioprotective effect of lycopene, a naturally occurring dietary carotenoid, on γ-radiation induced toxicity in cultured rat hepatocytes. The cellular changes were estimated using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), ceruloplasmin, vitamins A, E, C and uric acid. The DNA damage was analysed by single cell gel electrophoresis (comet assay). The increase in the severity of DNA damage was observed with the increase in γ-radiation dose (1, 2 and 4 Gy) in cultured rat hepatocytes. TBARS were increased significantly whereas the levels of GSH, vitamins C, E and A, ceruloplasmin, uric acid and antioxidant enzymes were significantly decreased in γ-irradiated groups. The maximum damage to hepatocytes was observed at 4 Gy irradiation. Pretreatment with lycopene (1.86, 9.31 and 18.62 μM) showed a significant decrease in the levels of TBARS and DNA damage. The antioxidant enzymes increased significantly along with the levels of GSH, vitamins A, E, C, uric acid and ceruloplasmin. The maximum protection of hepatocytes was observed at 9.31 μM of lycopene pretreatment. Thus, our results show that pretreatment with lycopene offers protection against γ-radiation induced cellular damage and can be developed as an effective radioprotector during radiotherapy.

Keywords: Lycopene; Radioprotector; γ-Radiation; Comet assay; Antioxidant; DNA damage

Complex oligosaccharides are N-linked to Kv3 voltage-gated K⁺ channels in rat brain by Tara A. Cartwright; Melissa J. Corey; Ruth A. Schwalbe (pp. 666-671).

Neuronal Kv3 voltage-gated K⁺ channels have two absolutely conserved N-glycosylation sites. Here, it is shown that Kv3.1, 3.3, and 3.4 channels are N-glycosylated in rat brain. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced faster migrating immunobands than those of undigested membranes. Additionally, partial PNGase F digests showed that both sites are occupied by oligosaccharides. Neuraminidase treatment produced a smaller immunoband shift relative to PNGase F treatment. These results indicate that both sites are highly available and occupied by N-linked oligosaccharides for Kv3.1, 3.3, and 3.4 in rat brain, and furthermore that at least one oligosaccharide is of complex type. Additionally, these results point to an extracytoplasmic S1–S2 linker in Kv3 proteins expressed in native membranes. We suggest that N-glycosylation processing of Kv3 channels is critical for the expression of K⁺ currents at the surface of neurons, and perhaps contributes to the pathophysiology of congenital disorders of glycosylation.

Keywords: Abbreviations; Kv; voltage-gated K; ⁺; channel; Endo H; Endoglycosidase H; CDG; Congenital disorders of glycosylation; PNGase F; peptide N glycosidase FGlycosylation; Topology; K; ⁺; channel; Brain; Congenital disorder of glycosylation

Connective tissue growth factor binds to fibronectin through the type I repeat modules and enhances the affinity of fibronectin to fibrin by Koji Yoshida; Hiroshi Munakata (pp. 672-680).

Connective tissue growth factor (CTGF) is a member of the CCN family of the cysteine-rich proteins and involved in wound healing and fibrosis. We have previously shown a biochemical interaction between the CTGF and fibronectin (FN) using the yeast two-hybrid system. In this study, we confirmed the interaction between the CTGF and FN using the surface plasmon resonance (SPR) and solid-phase binding analysis. Our results show that the regions containing the FN type I repeat modules (the N-terminal fibrin, the gelatin-collagen and the C-terminal fibrin binding domains) of FN and the C-terminal domain of CTGF are required for the interaction. We also demonstrated that CTGF enhances the affinity of FN to fibrin. It appears that CTGF contributes to the extracellular matrix accumulation in wound healing and tissue fibrosis by enhancing the affinity of FN to fibrin. Because CTGF is up-regulated during the tissue repair and in coagulation cascade-associated fibrotic disorders, the new function of CTGF found in this study is consistent with its physiological role.

Keywords: Abbreviations; CTGF; connective tissue growth factor; FN; fibronectin; BSA; bovine serum albumin; IGFBP; insulin-like growth factor binding protein; VWC; von Willebrand factor type C module; TSP1; thrombospondin type 1 repeat; CT; C-terminal module; GCBD; gelatin-collagen binding domain; CBD; cell binding domain; SPR; surface plasmon resonance; NFBD; N-terminal fibrin binding domain; CFBD; C-terminal fibrin binding domain; TGF-β; transforming growth factor-βConnective tissue growth factor; Fibronectin; Fibrin; Two-hybrid

Targeting potential and anti-HIV activity of lamivudine loaded mannosylated poly (propyleneimine) dendrimer by Tathagata Dutta; Narendra K. Jain (pp. 681-686).

T-lymphocytes, dendritic cells and macrophages are the target cells for HIV. The infected macrophages are considered as reservoirs for spreading the virus . Treatment of HIV infection therefore must reach these cells in addition to the organs like brain, liver and bone marrow. Lectin receptors, which act as molecular targets for sugar molecules, are found on the surface of these cells of the phagocytic system. The purpose of the present study is to investigate the targeting potential and anti HIV activity of lamivudine (3TC) loaded mannosylated fifth generation Poly (propyleneimine) dendrimers (MPPI). The entrapment efficiency of 3TC loaded MPPI and 5th generation poly(propyleneimine) dendrimer (PPI) were found to be 43.27±0.13% and 35.69±0.2% respectively. The in vitro drug release profile shows that while PPI releases the drug by 24 h, the MPPI slows down and hence prolongs the release up to 144 h (96.89±1.8% in case of MPPI). The results of in vitro ligand agglutination assay indicated that even after conjugation with PPI, mannose displayed binding specificity towards Con A. The subtoxic concentrations of free 3TC, blank PPI, blank MPPI, drug loaded PPI and drug loaded MPPI, determined on MT2 cells, were found to be 0.625, 0.039, 0.156, 0.039 and 0.156 nM/ml respectively. Significant increase in cellular uptake of 3TC was observed when MPPI was used, which was 21 and 8.3 times higher than that of free drug ( p<0.001) and PPI ( p<0.001) at 48 h respectively. Antiretroviral activity was determined using MT2 cell lines by estimating p24 antigen by ELISA. 3TC loaded PPI and MPPI formulations were found to possess higher anti-HIV activity at a concentration as low as 0.019 nM/ml, as compared to that of free drug, which was found to be extremely significant ( p<0.001). The significantly higher anti-HIV activity of PPI and MPPI is due to the enhanced cellular uptake of 3TC in formulation as compared to that of free drug Results suggest that the proposed carrier hold potential to increase the efficacy and reduce the toxicity of antiretroviral therapy.

Keywords: Poly (propyleneimine) dendrimer; Mannosylated dendrimer; Anti HIV activity; Macrophage Targeting; Lamivudine

Inhibition of PTEN and activation of Akt by menadione by Kyoko Yoshikawa; Kiyomi Nigorikawa; Mariko Tsukamoto; Namiko Tamura; Kaoru Hazeki; Osamu Hazeki (pp. 687-693).

Menadione (vitamin K₃) has been shown to activate Erk in several cell lines. This effect has been shown to be due to the activation of EGF receptors (EGFR) as a result of inhibition of some protein tyrosine phosphatases. In the present study, we examined the effects of menadione on Akt in Chinese hamster ovary cells. The phosphorylation of Akt by menadione was not inhibited by AG1478, an inhibitor of EGFR. Menadione inhibited the lipid phosphatase activity of PTEN in a cell-free system. In an intact cell system, menadione inhibited the effect of transfected PTEN on Akt. Thus, one mechanism of its action was considered the accelerated activation of Akt through inhibition of PTEN. This was not the sole mechanism responsible for the EGFR-independent activation of Akt, because menadione attenuated the rate of Akt dephosphorylation even in PTEN-null PC3 cells. The decelerated inactivation of Akt, probably through inhibition of some tyrosine phosphatases, was considered another mechanism of its action.

Keywords: Abbreviations; CHO-IR cells; Chinese hamster ovary cells expressing insulin receptors; IRβ; the β subunit of insulin receptor; PI 3-kinase; phosphoinositide 3-kinase; pNPP; p-nitrophenyl phosphate; PtdIns-3,4,5-P; ₃; phosphatidylinositol 3,4,5-triphosphate; PTEN; phosphatase and tensin homolog deleted on chromosome 10PTEN; Akt; Naphthoquinone; Menadione (vitamin K; ₃; )

Ontogeny of rdh9 (Crad3) expression: Ablation causes changes in retinoid and steroid metabolizing enzymes, but RXR and androgen signaling seem normal by Peirong Hu; Min Zhang; Joseph L. Napoli (pp. 694-705).

Crad3 ( cis-retinol/androgen dehydrogenase 3), a short-chain dehydrogenase/reductase, converts 9-cis-retinol into 9-cis-retinal and 3α-androstanediol into dihydrotestosterone. Crad3 may serve in biosynthesis of 9-cis-retinoic acid, a putative RXR ligand, and/or regeneration of potent androgens. RT-PCR showed that expression of the gene that encodes Crad3, rdh9, begins in liver by e11.5, and in kidney, testis, brain and intestine during e15.5–e16.5. In situ hybridization showed rdh9 expression in embryonic liver, ganglia, small intestine, lung, skin and vertebral cartilage. In adult, in situ hybridization revealed rdh9 expression intensely in hepatocytes, weakly in kidney glomerulus, and intensely in collecting tubules. In intestine, undifferentiated epithelia had greater expression than differentiated epithelia at the distal villus end. Testes expressed rdh9 in spermatogonia, and weakly in Leydig cells. Adult brain expressed rdh9 in the dentate gyrus and CA regions of the hippocampus, the cerebellum Purkinje cells, and the glomerular and mitral cell layers of the olfactory bulb. Rdh9-null mice, backcrossed against C57BL/6J mice, were born in Mendelian frequency, were healthy and fertile, and had normal tissue retinoid and serum dihydrotestosterone levels. Expression of rdh1, a gene that encodes an efficient retinol dehydrogenase, decreased 3- to 8-fold in rdh9-null mice, depending on dietary vitamin A. Microarray analysis and quantitative PCR revealed 2- to 4-fold increases in mRNA of enzymes that catalyze xenobiotic and steroid metabolism, including Cyp2, Cyp3, 11β-hydroxysteroid dehydrogenase type 2, and 17β-hydroxsteroid dehydrogenases types 4 and 5. These data indicate widespread Crad3 function(s) in steroid and/or retinoid metabolism starting mid embryogenesis.

Keywords: Abbreviations; Adh; alcohol dehydrogenase; Crad; cis; -retinoid androgen dehydrogenase; DHT; dihydrotestosterone; HPLC; high-performance liquid chromatography; HSD; hydroxysteroid dehydrogenase; Raldh; retinal dehydrogenase; RA; retinoic acid; RE; retinyl ester; SDR; short-chain dehydrogenase/reductaseRetinoic acid; Dihydrotestosterone; Short-chain dehydrogenase/reductase; Androgen; Hydroxysteroid dehydrogenase

Purification and kinetic properties of 6-phosphofructo-1-kinase from gilthead sea bream muscle by Dominica Mediavilla; Isidoro Metón; Isabel V. Baanante (pp. 706-715).

The kinetic properties of 6-phosphofructo-1-kinase (PFK) from skeletal muscle (PFKM) of gilthead sea bream ( Sparus aurata) were studied, after 10,900-fold purification to homogeneity. The native enzyme had an apparent molecular mass of 662 kDa and is composed of 81 kDa subunits, suggesting a homooctameric structure. At physiological pH, S. aurata PFKM exhibited sigmoidal kinetics for the substrates, fructose-6-phosphate (fru-6-P) and ATP. Fructose-2,6-bisphosphate (fru-2,6-P₂) converted the saturation curves for fru-6-P to hyperbolic, activated PFKM synergistically with other positive effectors of the enzyme such as AMP and ADP, and counteracted ATP and citrate inhibition. The fish enzyme showed differences regarding other animal PFKs: it is active as a homooctamer, and fru-2,6-P₂ and pH affected affinity for ATP. By monitoring incorporation of³²P from ATP, we show that fish PFKM is a substrate for the cAMP-dependent protein kinase. The mechanism involved in PFKM activation by phosphorylation contrasts with previous observations in other species: it increased V_max and did not affect affinity for fru-6-P. Unlike the mammalian muscle enzyme, our findings support that phosphorylation of PFKM may exert a major role during starvation in fish muscle.

Keywords: 6-Phosphofructo-1-kinase; Glycolysis; Kinetics; Skeletal muscle; Sparus aurata

Mutanase from a Paenibacillus isolate: Nucleotide sequence of the gene and properties of recombinant enzymes by Nobuyuki Sumitomo; Katsuhisa Saeki; Katsuya Ozaki; Susumu Ito; Tohru Kobayashi (pp. 716-724).

A mutanase (α-1,3-glucanase)-producing microorganism was isolated from a soil sample and was identified as a relative of Paenibacillus sp. The mutanase was purified to homogeneity from culture, and its molecular mass was around 57 kDa. The gene for the mutanase was cloned by PCR using primers based on the N-terminal amino acid sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of 3651-bp open reading frame that encoded a predicted 1217-amino acid polypeptide including a 43-amino acid signal peptide. The mature enzyme showed similarity to mutanases RM1 of Bacillus sp. strain RM1 and KA-304 of Bacillus circulans with 65.6% and 62.7% identity, respectively. The predicted molecular mass of the mutanase was 123 kDa. Thus, the enzyme purified from the isolate appears to be truncated by proteolysis. The genes for the full-length and truncated mutanases were expressed in Bacillus subtilis cells, and the corresponding recombinant enzymes were purified to homogeneity. The molecular masses of the two enzymes were 116 and 57 kDa, respectively. The specific activity was 10-fold higher for the full-length enzyme than for the truncated enzyme. The optimal pH and temperature for both recombinant enzymes was pH 6.4 in citrate buffer and 45 °C to 50 °C. Amongst several tested polysaccharides, the recombinant full-length enzyme specifically hydrolyzed mutan.

Keywords: Mutanase; α-1,3 glucanase; Cloning; Mutan; Paenibacillus

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