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BBA - General Subjects (v.1760, #11)

Editorial Board (pp. ii).

Effects of rhDecorin on TGF-β1 induced human hepatic stellate cells LX-2 activation by Yue-Feng Shi; Qi Zhang; Pik-Yuen Cheung; Lin Shi; Chi-Chun Fong; Yaou Zhang; Chi-Hung Tzang; Bernard P.L. Chan; Wang-Fun Fong; Jay Chun; Hsiang-fu Kung; Mengsu Yang (pp. 1587-1595).

Decorin is a small leucine-rich extracellular matrix proteoglycan composed of a core protein with a single glycosaminoglycan (GAG) chain near the N-terminus and N-glycosylated at three potential sites. Decorin is involved in the regulation of formation and organization of collagen fibrils, modulation of the activity of growth factors such as transforming growth factor β (TGF-β), and exerts other effects on cell proliferation and behavior. Increasing evidences show that decorin plays an important role in fibrogenesis by regulating TGF-β, a key stimulator of fibrosis, and by directly modulating the degradation of extracellular matrix (ECM) from activated hepatic stellate cells (HSCs). In this study, the core protein of human decorin was cloned and expressed in Escherichia coli. The purified recombinant human decorin (rhDecorin) significantly inhibited the proliferation of LX-2 cells, a human HSC cell line, stimulated by TGF-β1. RT-PCR result showed that the expression of metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were reduced by rhDecorin in LX-2 cells stimulated by TGF-β1. Furthermore, the protein expression of smooth muscle-α-actin (α-SMA), collagen type III and phosphorylated Smad2 (p-Smad2) was significantly decreased in the presence of rhDecorin. rhDecorin also reduced fibrillogenesis of collagen type I in a dose-dependent manner. Gene expression profiles of LX-2 cells stimulated by TGF-β1 in the presence and the absence of rhDecorin were obtained by using cDNA microarray technique and differentially expressed genes were identified to provide further insight into the molecular action mechanism of decorin on LX-2 cells.

Keywords: Abbreviations; HSCs; hepatic stellate cells; TGF-β1; transforming growth factor-β1; ECM; extracellular matrix; MMP; metalloproteinase; TIMP; tissue inhibitor of metalloproteinase; α-SMA; smooth muscle-α-actinDecorin; Hepatic stellate cell; LX-2 cell line; Fibrogenesis; Liver fibrosis

Lipid peroxidation as a source of oxidative damage in Helicobacter pylori: Protective roles of peroxiredoxins by Ge Wang; Yang Hong; Michael K. Johnson; Robert J. Maier (pp. 1596-1603).

Oxidative stress conditions lead to enzymatic and non-enzymatic unsaturated fatty acid-initiated lipid peroxidation reactions. One exacerbating product is lipid hydroperoxide (LOOH) which itself promotes formation of several additional peroxyl radicals. Helicobacter pylori mutant strains with disruptions in genes encoding the peroxiredoxins, alkyl hydroperoxide reductase ( ahpC) and the bacterioferritin comigratory protein ( bcp), were more sensitive than the parent strain to oxidizing agents. These mutant strains were particularly sensitive, compared to the wild type, to killing by the unsaturated fatty acid linolenic acid but were not sensitive to the saturated fatty acid palmitic acid. A double mutant strain ( ahpC bcp) accumulated more than 3-fold more lipid peroxides than the parent strain, indicating these peroxiredoxins together play a role in detoxifying lipid peroxides. The level of free iron accumulation, a signature of oxidative stress damage, was correlated specifically to organic peroxide-mediated stress by both in vivo and in vitro approaches. Free iron accumulation and concomitant destruction of [Fe–S] cluster-containing proteins (hydrogenase and aconitase) was correlated to damage mediated by exogenous t-butyl peroxide, or separately to intracellular accumulation of lipid peroxides in mutant strains. A major macromolecular target of accumulating lipid peroxides in H. pylori is DNA, as mutant analysis approaches combined with quantitative DNA fragmentation studies and specific DNA damage assessment (i.e. 8-oxoguanine formation) were used to demonstrate that such damage was especially associated with ahpC and ahpC bcp strains.

Keywords: Helicobacter; Lipid peroxidation; Peroxiredoxins; Oxidative DNA damage; Intracellular free iron; [Fe–S] cluster proteins

An insight into the gene delivery mechanism of the arginine peptide system: Role of the peptide/DNA complex size by Hong Seok Choi; Hyun Hee Kim; Jai Myung Yang; Sungho Shin (pp. 1604-1612).

Cationic peptides have been used successfully to transfer macromolecules into living cells. Previously, we have reported a short arginine peptide-based gene delivery system. However, the mechanisms that allow arginine peptides to promote gene delivery yet remain unknown. In the present study, we investigated the effect of the arginine peptide/DNA complex size on the transfection efficiency. After combining peptides with DNA, a 400 nm complex was observed. As the incubation time was increased, the complex grew larger, reaching 6 μm after 1 h of incubation. Transfection and cellular uptake efficiency were likewise investigated for the effects of the different sizes of complexes. Large complexes were found to be advantageous for transfection. However, better internalization efficiency was found with small complexes, indicating that the amount of peptide/DNA complexes taken up by cells is not the rate-limiting step in the final transfection efficiency. The intracellular path of the peptide/DNA complex was studied using fluorescent labeling and confocal microscopy. In the early stages of transfection, complexes were observed only on the cell surface, and these complexes migrated into cytoplasm however, after 6 h, the presence of complexes in the perinuclear region was noted. We were able to detect colocalization of green and red fluorescence in both the cytoplasm and the nucleus. These results suggest that peptide/DNA complexes reach the nucleus as associated complexes.

Keywords: Arginine peptide; Complex size; Gene delivery; Intracellular tracking

Interleukin-15 increases glucose uptake in skeletal muscle An antidiabetogenic effect of the cytokine by Sílvia Busquets; Maite Figueras; Vanessa Almendro; Francisco J. López-Soriano; Josep M. Argilés (pp. 1613-1617).

Previous studies have demonstrated that interleukin-15 (IL-15) has important anabolic effects on muscle protein metabolism. In the present investigation we have analysed the effects of IL-15 on glucose metabolism in skeletal muscle. Administration of a single dose of the cytokine (100 μg/kg body weight) resulted in a 32% increase on glucose uptake (as measured by the uptake of 2-deoxyglucose) in skeletal muscle. The effects observed on glucose uptake were direct since in vitro incubations of rat EDL muscles in the presence of the cytokine resulted in a 30% increase in glucose uptake. Similarly, IL-15 increased glucose uptake in C2C12 cell cultures, this being related with an increase in both glucose oxidation to CO₂ and the incorporation into muscle lipid. The effects of the cytokine were associated with an increase in GLUT-4 mRNA, suggesting a higher effect in insulin sensitivity. In conclusion, the data presented here indicate that IL-15 facilitates glucose metabolism in skeletal muscle and, therefore, a possible role of the cytokine as an antidiabetogenic drug merits future investigations.

Keywords: Interleukin-15; Skeletal muscle; Glucose uptake; Glucose metabolism

Cyclic GMP-dependent protein kinase and soluble guanylyl cyclase disappear in elicited rat neutrophils by Małgorzata Ciuman; Jakub Siednienko; Rafał Czyżyk; Hanna Witwicka; Ewa Kołosionek; Marcin Kobiałka; Wojciech A. Gorczyca (pp. 1618-1623).

The nitric oxide/soluble guanylyl cyclase/cGMP-dependent protein kinase (NO/sGC/PKG) cascade has been shown to affect important functions of circulating neutrophils. We demonstrate that neutrophils isolated from rats treated intraperitoneally with peptone protease cannot use this signaling pathway. Although PKG was detected at both the mRNA and protein levels in peripheral blood neutrophils (PBNs) of control rats, it was expressed neither in PBNs nor in peritoneal exudate neutrophils (PENs) of provoked rats. Also, mRNA of the α and β chains of heterodimeric sGC was present in PBNs, but absent in PENs. Consistently, PBNs responded to activators of sGC with cGMP synthesis, while PENs did not. These results showed that neutrophils recruited by a provoking agent lost PKG and, in the case of PENs, also sGC and thus the capacity to respond to NO with cGMP signaling. We speculate that such downregulation of the sGC/PKG pathway is likely a result of the high activity of inducible NO synthase observed in inflammatory neutrophils.

Keywords: Guanylyl cyclase; Protein kinase; Cyclic GMP; Nitric oxide; Neutrophil

C-phycocyanin transcriptionally regulates uPA mRNA through cAMP mediated PKA pathway in human fibroblast WI-38 cells by H.K. Madhyastha; K.S. Radha; M. Sugiki; S. Omura; M. Maruyama (pp. 1624-1630).

We have previously demonstrated the efficacy of c-phycocyanin in up-regulation of urokinase-type plasminogen activator (uPA) in bovine endothelial cell line. However, the mechanism of action and pathway elucidation in uPA regulation is unclear. In experiments reported here, we have investigated the mechanism of action of c-phycocyanin (c-pc) induced uPA gene modulation in human fibroblast (WI-38) cell line. ELISA test confirmed that c-pc increased the uPA antigen whereas PAI-1 antigen level was unaffected. Treatment of cells with c-pc significantly ( P<0.05) enhanced the uPA mRNA level in a dose (50 μg/ml) and time dependent (up to 4 h) manner. This effect of c-pc was abolished by treatment with dichloro-1-β-d-ribofuranosyl benzamidazole (DRB) (10 μg/ml). Co-treatment of c-pc with 200 μg/ml cycloheximide (CHX), translation inhibitor, resulted in over accumulation of uPA mRNA. These results suggest that uPA induction by c-pc is transcriptionally regulated and does not require de novo protein synthesis. We also provide evidence that c-pc stimulates uPA gene through cAMP dependent pathway as adenylyl cyclase (AC) inhibitor, dideoxyadenosine (DDA) significantly inhibited the uPA mRNA expression and co-treatment with adenylyl cyclase analogue, dBcAMP recovered the effect of c-pc on gene activity. Furthermore, the present investigation provides evidence on the regulatory pathway involved in the c-pc stimulus. C-pc induced uPA expression was completely inhibited by PKA inhibitor (KT 5200), indicating the regulation is dependent on PKA pathway. Elimination of PKC pathway components by prolonged incubation with excess amount of phorbol 12-myristate 13-acetate (PMA) failed to abolish the c-pc effect on uPA expression indicating the regulation is independent of PKC pathway. Taken together, our data indicate that uPA gene regulation by c-pc is transcriptionally controlled through cAMP mediated PKA pathway.

Keywords: Abbreviations; c-pc; c-phycocyanin; uPA; urokinase-type plasminogen activator; CHX; cycloheximide; DRB; dichloro-1-β-; d; -ribofuranosyl benzamidazole; PMA; phorbol 12-myristate 13-acetate; DDA; dideoxyadenosine; dBcAMP; dibutyryl cyclicAMP Spirulina fusiformis; C-phycocyanin; uPA; Fibroblast; PKA pathway

A remodeling system for the oligosaccharide chains on glycoproteins with microbial endo-β- N-acetylglucosaminidases by Kiyotaka Fujita; Kenji Yamamoto (pp. 1631-1635).

Endo-M, endo-β- N-acetylglucosaminidase from Mucor hiemalis, transferred the complex type oligosaccharide of sialoglycopeptide to partially deglycosylated proteins ( N-acetylglucosamine-attached proteins), which were prepared by excluding high-mannose type oligosaccharides from glycoproteins with Endo-H, endo-β- N-acetylglucosaminidase from Streptomyces plicatus. This finding indicated that the high-mannose type oligosaccharides on glycoproteins can be changed to complex type ones by the transglycosylation activity of Endo-M. This is the first report of the establishment of a remodeling system for the different types of oligosaccharides on glycoproteins with microbial endo-β- N-acetylglucosaminidases having different substrate specificities. Endo-M is a powerful tool for the in vitro synthesis of glycoproteins containing complex type oligosaccharides from glycoproteins produced by yeast.

Keywords: Abbreviations; CPY; carboxypeptidase Y; RNase; ribonuclease; HRP; horseradish peroxidase; Con A; concanavalin A; SSA; Sambucus sieboldiana; agglutinin; SGP; sialoglycopeptideNeoglycoprotein; Endo-β-; N; -acetylglucosaminidase; Transglycosylation

α,β-dicarbonyl reduction by Saccharomycesd-arabinose dehydrogenase by Barry van Bergen; Rona Strasser; Normand Cyr; John D. Sheppard; Armando Jardim (pp. 1636-1645).

An α,β-dicarbonyl reductase activity was purified from Saccharomyces cerevisiae and identified as the cytosolic enzymed-Arabinose dehydrogenase ( ARA1) by MALDI-TOF/TOF. Size exclusion chromatography analysis of recombinant Ara1p revealed that this protein formed a homodimer. Ara1p catalyzed the reduction of the reactive α,β-dicarbonyl compounds methylglyoxal, diacetyl, and pentanedione in a NADPH dependant manner. Ara1p had apparent K_m values of ∼14 mM, 7 mM and 4 mM for methylglyoxal, diacetyl and pentanedione respectively, with corresponding turnover rates of 4.4, 6.9 and 5.9 s−¹ at pH 7.0. pH profiling showed that Ara1p had a pH optimum of 4.5 for the diacetyl reduction reaction. Ara1p also catalyzed the NADP⁺ dependant oxidation of acetoin; however this back reaction only occurred at alkaline pH values. That Ara1p was important for degradation of α,β-dicarbonyl substrates was further supported by the observation that ara1-Δ knockout yeast mutants exhibited a decreased growth rate phenotype in media containing diacetyl.

Keywords: d; -Arabinose; ARA1; Dehydrogenase; Dicarbonyl; Diacetyl; ARA1p

Characterization of prmt7α and β isozymes from Chinese hamster cells sensitive and resistant to topoisomerase II inhibitors by Laurent Gros; Axelle Renodon-Cornière; Bruno Robert de Saint Vincent; Marcin Feder; Janusz M. Bujnicki; Alain Jacquemin-Sablon (pp. 1646-1656).

By selection of genetic suppressor elements (GSEs) conferring resistance to topoisomerase II inhibitors in Chinese hamster cells (DC-3F), we identified a gene encoding two proteins of 78 and 82 kDa which belong to the protein arginine methyltransferase (PRMT) family. Down-regulation of these enzymes (named PRMT7α and β), either induced by an antisense GSE or as observed in the 9-OH-ellipticine (9-OH-E) resistant mutant DC-3F/9-OH-E, was responsible for cell resistance to various DNA damaging agents. Alternative splicing alterations in the 5'-terminal region and changes of the polyadenylation site of PRMT7 mRNAs were observed in these resistant mutant cells. PRMT7α and β are isoforms of a highly conserved protein containing two copies of a module common to all PRMTs, comprising a Rossmann-fold domain and a β-barrel domain. The C-terminal repeat appears to be degenerate and catalytically inactive. PRMT7α and β form homo- and hetero-dimers but differ by their sub-cellular localization and in vitro recognize different substrates. PRMT7β was only observed in Chinese hamster cells while mouse 10T1/2 fibroblasts only contain PRMT7α. Surprisingly, in human cells the anti-PRMT7 antibody essentially recognized an ∼37 kDa peptide, which is not formed during extraction, and a faint band at 78 kDa. Analysis of in vitro and in vivo methylation patterns in cell lines under- or over-expressing PRMT7α and β detected a discrete number of proteins which methylation and/or expression are under the control of these enzymes.

Keywords: Protein arginine methyltransferases; PRTMT7α and β; Anticancer drug resistance

Resveratrol exerts its antiproliferative effect on HepG2 hepatocellular carcinoma cells, by inducing cell cycle arrest, and NOS activation by George Notas; Artemissia-Phoebe Nifli; Marilena Kampa; Joseph Vercauteren; Elias Kouroumalis; Elias Castanas (pp. 1657-1666).

The stilbene resveratrol exerts antiproliferative and proapoptotic actions on a number of different cancer cell lines, through diverse mechanisms, including antioxidant effects, enzyme, growth factor and hormone receptor binding, and nucleic acid direct or indirect interactions. Although resveratrol accumulates in the liver, its effect on hepatocellular carcinoma has not been extensively studied. We have used the human hepatocyte-derived cancer cell line HepG2 to address the possible action of resveratrol on cell growth and to examine some possible mechanisms of action. Our results indicate that the stilbene inhibits potently cell proliferation, reduces the production of reactive oxygen species and induces apoptosis, through cell cycle arrest in G₁ and G₂/M phases. Furthermore it modulates the NO/NOS system, by increasing iNOS and eNOS expression, NOS activity and NO production. Inhibition of NOS enzymes attenuates its antiproliferative effect. These data could be of value in possible prevention or adjuvant treatment of hepatocellular carcinoma, through an increased consumption of resveratrol-rich foods and beverages.

Keywords: Resveratrol; Hepatocellular carcinoma; Cell cycle; Apoptosis; Nitric oxide (NO); Nitric oxide synthase (NOS)

A newd-2-hydroxyacid dehydrogenase with dual coenzyme-specificity from Haloferax mediterranei, sequence analysis and heterologous overexpression by J. Domenech; J. Ferrer (pp. 1667-1674).

A gene encoding a newd-2-hydroxyacid dehydrogenase (E.C. 1.1.1.) from the halophilic Archaeon Haloferax mediterranei has been sequenced, cloned and expressed in Escherichia coli cells with the inducible expression plasmid pET3a. The nucleotide sequence analysis showed an open reading frame of 927 bp which encodes a 308 amino acid protein. Multiple amino acid sequence alignments of the D-2-hydroxyacid dehydrogenase from H. mediterranei showed high homology with D-2-hydroxyacid dehydrogenases from different organisms and other enzymes of this family. Analysis of the amino acid sequence showed catalytic residues conserved in hydroxyacid dehydrogenases withd-stereospecificity. In the reductive reaction, the enzyme showed broad substrate specificity, although α-ketoisoleucine was the most favourable of all α-ketocarboxylic acids tested. Kinetic data revealed that this new D-2-hydroxyacid dehydrogenase from H. mediterranei exhibits dual coenzyme-specificity, using both NADPH and NADH as coenzymes. To date, all D-2-hydroxyacid dehydrogenases have been found to be NADH-dependent. Here, we report the first example of a D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity.

Keywords: D-2-hydroxyacid dehydrogenase; inclusion bodies; renaturalization; dual coenzyme-specificity; α-ketocarboxylic acid

Expression and polysome association of YB-1 in various tissues at different stages in the lifespan of mice by Arisa Miwa; Takashi Higuchi; Shunsuke Kobayashi (pp. 1675-1681).

Tissue-specific translational regulation is important for gene expression. YB-1 binds to mRNAs to form mRNPs and affects translation. In this study we investigated expression and polysome association of YB-1 in various tissues at different stages in the lifespan of mice. YB-1 levels decreased markedly with growth in brain, heart and muscle, but increased in the spleen. In lung, kidney and testis, the levels of YB-1 diminished with aging. In liver, no significant change in the level of YB-1 was observed throughout life. We further showed that the distribution pattern of YB-1 on a sucrose gradient differed according to tissue. Moreover, the distribution pattern of YB-1 changed drastically with growth in the liver. In 5-day-old liver, YB-1 was distributed almost exclusively in nonpolysomal fractions, whereas in 4-week-old liver, it was associated with heavy-sedimenting polysomes, as was the case in 5-day-old brain. Immunohistochemical analysis revealed that YB-1 is mainly a cytoplasmic protein in these tissues. Our results indicate that the expression and polysome association of YB-1 are regulated with growth or aging in a tissue-specific manner, presumably to control gene expression at the translational level in each tissue.

Keywords: Y-box protein; Expression; Polysome association; Tissue-specific manner; Differential stages in the lifespan

Berberine-stimulated glucose uptake in L6 myotubes involves both AMPK and p38 MAPK by Zhe Cheng; Tao Pang; Min Gu; An-Hui Gao; Chuan-Ming Xie; Jing-Ya Li; Fa-Jun Nan; Jia Li (pp. 1682-1689).

Berberine is a plant alkaloid used in traditional Chinese medicine and has been reported to have antihyperglycemic activity in NIDDM patients. However, the molecular basis for this action is yet to be elucidated. Here we investigate the effects and signaling pathways of berberine on L6 rat skeletal muscles. Our study demonstrates that berberine stimulates glucose uptake in a time- and dose-dependent manner. Intriguingly, berberine-stimulated glucose uptake does not vary as insulin concentration increases, and could not be blocked by the PI 3-kinase inhibitor wortmannin. Berberine only weakly stimulates the phosphorylation of Akt/PKB, a key molecule in the insulin signaling pathway, but strongly promotes the phosphorylation of AMPK and p38 MAPK. The effects of berberine are not a result of pro-oxidant action, but a consequence of an increased cellular AMP:ATP ratio. Moreover, berberine-stimulated glucose uptake is inhibited by the AMPK inhibitor Compound C and the p38 MAPK inhibitor SB202190. Inhibition of AMPK reduces p38 MAPK phosphorylation, suggesting that AMPK lies upstream of p38 MAPK. These results suggest that berberine circumvents insulin signaling pathways and stimulates glucose uptake through the AMP-AMPK-p38 MAPK pathway, which may account for the antihyperglycemic effects of this drug.

Keywords: Abbreviations; 2-DOG; 2-deoxyglucose; ACC; acetyl-CoA carboxylase; AICAR; 5′-amino-4-imidazolecarboxamide-riboside; AMPK; AMP-activated protein kinase; ATCC; American Type Culture Collection; ECL; enhanced chemiluminescence; FBS; fetal bovine serum; HG-DMEM; high glucose Dulbecco's modified Eagle medium; IRS-1; insulin receptor substrate-1; NAC; N; -acetyl-; l; -cysteine; NIDDM; non-insulin-dependent diabetes mellitus; P38 MAPK; p38 mitogen-activated protein kinase; PI 3-kinase; phosphatidylinositol 3-kinase; PVDF; polyvinylidene difluorideBerberine; Glucose uptake; AMPK; p38 MAPK; Myotube

Quenching of superoxide radicals by green fluorescent protein by Fadi Bou-Abdallah; N. Dennis Chasteen; Michael P. Lesser (pp. 1690-1695).

Green fluorescent proteins (GFP) are widely used in vivo molecular markers. These proteins are particularly resistant, and maintain function, under a variety of cellular conditions such as pH extremes and elevated temperatures. Green fluorescent proteins are also abundant in several groups of marine invertebrates including reef-forming corals. While molecular oxygen is required for the post-translational maturation of the protein, mature GFPs are found in corals where hyperoxia and reactive oxygen species (ROS) occur due to the photosynthetic activity of algal symbionts. In vitro spin trapping electron paramagnetic resonance and spectrophotometric assays of superoxide dismutase (SOD)-like enzyme activity show that wild type GFP from the hydromedusa, Aequorea victoria, quenches superoxide radicals (O₂⁻) and exhibits SOD-like activity by competing with cytochrome c for reaction with O₂⁻. When exposed to high amounts of O₂⁻ the SOD-like activity and protein structure of GFP are altered without significant changes to the fluorescent properties of the protein. Because of the distribution of fluorescent proteins in both the epithelial and gastrodermal cells of reef-forming corals we propose that GFP, and possibly other fluorescent proteins, can provide supplementary antioxidant protection.

Keywords: GFP; Superoxide radical; Coral; EPR; Spin trapping; Capillary electrophoresis

On the simulation of enzymatic digest patterns: The fragmentation of oligomeric and polymeric galacturonides by endo-polygalacturonase II by Jonathan J. Hunt; Randall Cameron; Martin A.K. Williams (pp. 1696-1703).

A simulation methodology for predicting the time-course of enzymatic digestions is described. The model is based solely on the enzyme's subsite architecture and concomitant binding energies. This allows subsite binding energies to be used to predict the evolution of the relative amounts of different products during the digestion of arbitrary mixtures of oligomeric or polymeric substrates. The methodology has been specifically demonstrated by studying the fragmentation of a population of oligogalacturonides of varying degrees of polymerization, when digested by endo-polygalacturonase II (endo-PG II) from Aspergillus niger.

Keywords: Enzyme; Polysaccharide; Endo-polygalacturonase; Oligogalacturonides; Oligomeric and polymeric substrate

Selective binding of imatinib to the genetic variants of human α₁-acid glycoprotein by Ilona Fitos; Júlia Visy; Ferenc Zsila; György Mády; Miklós Simonyi (pp. 1704-1712).

Imatinib is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia. Its strong plasma protein binding referred to α₁-acid glycoprotein (AGP) component was found to inhibit the pharmacological activity. AGP shows genetic polymorphism and the two main genetic variants have different drug binding properties. The binding characteristics of imatinib to AGP genetic variants and the possibility of its binding interactions were investigated by various methods. The results proved that binding of imatinib to the two main genetic variants is very different, the high affinity binding belongs dominantly to the F1-S variant. This interaction is accompanied with specific spectral changes (induced circular dichroism, UV change, intrinsic fluorescence quenching), suggesting that the bound ligand has chiral conformation that would largely overlap with other ligands inside the protein cavity. Binding parameters of K_a=1.7(±0.2)×10⁶M−¹ and n=0.94 could be determined for the binding on the F1-S variant at 37°. Imatinib binding on the A variant is weaker and less specific. The binding affinity of imatinib to human serum albumin (n K_a≈3×10⁴M−¹) is low. Pharmacologically relevant binding interactions with other drugs can be expected on the F1-S variant of AGP.

Keywords: Abbreviations; AGP; human α; ₁; -acid glycoprotein; CD; circular dichroism; TNS; p-(2-toluidino)-6-naphthalinsulfonate; AO; acridine orange; AODB; acridine orange-10-dodecyl bromideα; ₁; -Acid glycoprotein; Genetic variants; Imatinib; Induced circular dichroism; Fluorescence quenching; Drug binding interaction

Purification, properties and cDNA cloning of neoverrucotoxin (neoVTX), a hemolytic lethal factor from the stonefish Synanceia verrucosa venom by Atsushi Ueda; Mika Suzuki; Tomohiro Honma; Hiroshi Nagai; Yuji Nagashima; Kazuo Shiomi (pp. 1713-1722).

A proteinaceous toxin with hemolytic and lethal activities, named neoverrucotoxin (neoVTX), was purified from the venom fluid of stonefish Synanceia verrucosa and its primary structure was elucidated by a cDNA cloning technique. NeoVTX is a dimeric 166 kDa protein composed of α-subunit (702 amino acid residues) and β-subunit (699 amino acid residues) and lacks carbohydrate moieties. Its hemolytic activity is inhibited by anionic lipids, especially potently by cardiolipin. These properties are comparable to those of stonustoxin (SNTX) previously purified from S. horrida. Alignment of the amino acid sequences also reveals that the neoVTX α- and β-subunits share as high as 87 and 95% sequence identity with the SNTX α- and β-subunits, respectively. The distinct differences between neoVTX and SNTX are recognized only in the numbers of Cys residues (18 for neoVTX and 15 for SNTX) and free thiol groups (10 for neoVTX and 5 for SNTX). In contrast, neoVTX considerably differs from verrucotoxin (VTX), a tetrameric 322 kDa glycoprotein, previously purified from S. verrucosa. In addition, the sequence identity of the neoVTX β-subunit with the reported VTX β-subunit is 90%, being lower than that with the SNTX β-subunit.

Keywords: cDNA cloning; Neoverrucotoxin; Stonefish; Synanceia verrucosa; Toxin

NMR-based metabolic profiling of human hepatoma cells in relation to cell growth by culture media analysis by Alberta Tomassini Miccheli; Alfredo Miccheli; Roberta Di Clemente; Mariacristina Valerio; Pierpaolo Coluccia; Mariano Bizzarri; Filippo Conti (pp. 1723-1731).

Metabolic profiling is a metabolomic approach that allows the characterization of metabolic phenotypes under specific set of conditions. In the present paper we investigated the metabolism of sparse and high density cultures in relation to different cell growth phases. Changes in the metabolome were evaluated by using¹H-NMR spectroscopy, correlation map and Multivariate Data Analysis on the net balances of metabolites in the medium. This approach allowed us to identify two different metabolic profiles in relation to the cell growth phases in subconfluence and confluence cultures. The results have been interpreted on the basis of patterns of correlations obtained in the two physiological cell states. Cells almost arrested in G0/G1 phase by contact dependent growth inhibition underwent changes in the channeling of amino acids utilization from synthetic to energetic purpose and in anaplerosis/cataplerosis regulation of the TCA cycle.

Keywords: Metabolomics; ¹; H NMR; Cancer cell; Amino acid metabolism; HepG2

Dual mode of action of Bac7, a proline-rich antibacterial peptide by Elena Podda; Monica Benincasa; Sabrina Pacor; Fulvio Micali; Maura Mattiuzzo; Renato Gennaro; Marco Scocchi (pp. 1732-1740).

Proline-rich peptides are a unique group of antimicrobial peptides that exert their activity selectively against Gram-negative bacteria through an apparently non-membranolytic mode of action that is not yet well understood. We have investigated the mechanism underlying the antibacterial activity of the proline-rich cathelicidin Bac7 against Salmonella enterica and Escherichia coli. The killing and membrane permeabilization kinetics as well as the cellular localization were assessed for the fully active N-terminal fragment Bac7(1–35), its all-D enantiomer and for differentially active shortened fragments. At sub-micromolar concentrations, Bac7(1–35) rapidly killed bacteria by a non-lytic, energy-dependent mechanism, whereas its D-enantiomer was inactive. Furthermore, while theL-enantiomer was rapidly internalized into bacterial cells, the D-enantiomer was virtually excluded. At higher concentrations (≥64 μM), both L- and D-Bac7(1–35) were instead able to kill bacteria also via a lytic mechanism. Overall, these results suggest that Bac7 may inactivate bacteria via two different modes of action depending on its concentration: (i) at near-MIC concentrations via a mechanism based on a stereospecificity-dependent uptake that is likely followed by its binding to an intracellular target, and (ii) at concentrations several times the MIC value, via a non-stereoselective, membranolytic mechanism.

Keywords: Abbreviations; AMP; Antimicrobial peptide; CCCP; Carbonyl cyanide m-chlorophenylhydrazone; DNP; 2,4-dinitrophenol; MH; Mueller–Hinton; MIC; Minimum inhibitory concentration; PEG-PS; Polyethylene glycol-polystyrene; PI; Propidium iodide; TEM; Transmission electron microscopy; TBS; Tris-buffered saline solutionBactenecin 7; Proline-rich peptide; Cathelicidin peptide; Antibacterial activity; Mechanism of action; Membrane permeabilization

Pyrroloquinoline quinone nutritional status alters lysine metabolism and modulates mitochondrial DNA content in the mouse and rat by K.A. Bauerly; D.H. Storms; C.B. Harris; S. Hajizadeh; M.Y. Sun; C.P. Cheung; M.A. Satre; A.J. Fascetti; E. Tchaparian; R.B. Rucker (pp. 1741-1748).

Pyrroloquinoline quinone (PQQ) added to purified diets devoid of PQQ improves indices of perinatal development in rats and mice. Herein, PQQ nutritional status and lysine metabolism are described, prompted by a report that PQQ functions as a vitamin-like enzymatic cofactor important in lysine metabolism (Nature 422 [2003] 832). Alternatively, we propose that PQQ influences lysine metabolism, but by mechanisms that more likely involve changes in mitochondrial content. PQQ deprivation in both rats and mice resulted in a decrease in mitochondrial content. In rats, α-aminoadipic acid (αAA), which is derived from α-aminoadipic semialdehyde (αAAS) and made from lysine in mitochondria, and the plasma levels of amino acids known to be oxidized in mitochondria (e.g., Thr, Ser, and Gly) were correlated with changes in the liver mitochondrial content of PQQ-deprived rats, but not PQQ-supplemented rats. In contrast, the levels of NAD dependent α-aminoadipate-δ-semialdehyde dehydrogenase (AASDH), a cytosolic enzyme important to αAA production from αAAS, was not influenced by PQQ dietary status. Moreover, the levels of U26 mRNA were not significantly changed even when diets differed markedly in PQQ and dietary lysine content. U26 mRNA levels were measured, because of U26's proposed, albeit questionable role as a PQQ-dependent enzyme involved in αAA formation.

Keywords: Abbreviations; (αAA); α-aminoadipic acid or α-aminoadipate; (Lys2); α-aminoadipic acid reductase; (αAAS); α-aminoadipic-δ-semialdehyde; (DH); dehydrogenase; (PC); Δ; ¹; -piperidine-δ-carboxylate; (PQQ); pyrroloquinoline quinone; (U26); putative mouse homolog of αAA reductasePyrroloquinoline quinone; Lysine metabolism; α-aminoadipic-δ-semialdehyde dehydrogenase; Mitochondria

Glycated albumin (Amadori product) induces activation of MAP kinases in monocyte-like MonoMac 6 cells by Rowena Brandt; Sven Krantz (pp. 1749-1753).

Increased levels of glycated, Amadori-modified albumin are a risk factor for diabetic vascular disorders. Glycated albumin binds to specific receptors and induces cellular signaling pathways, the complexity of which is largely unknown. Binding of glycated albumin to MonoMac 6 cells leads to an activation of MAPK p44/42 (ERK1/2) and p38 with subsequent translocation of NF-κB into the nucleus. The activation of MAPK is in part mediated by protein kinase C activation, but a PKC-independent pathway via MEK-1 is also involved. Protein tyrosine kinases do not play a role in the activation of NF-κB. The results may have pathophysiological significance, because the MonoMac 6 cell line is not greatly different from blood monocytes.

Keywords: Abbreviations; AGEs; advanced glycation end products; AP-1; activator protein-1; ERK; extracellular signal-regulated kinase; GA; glycated albumin; IL-1β; interleukin 1β; MAPK; mitogen-activated protein kinases; NA; native, nonglycated albumin; NF-κB; nuclear factor-κB; PKC; protein kinase C; PTK; protein tyrosine kinase; ROS; reactive oxygen species; SDS-PAGE; sodium dodecylsulfate polyacrylamide gel electrophoresis; TNF; tumor necrosis factorGlycated albumin; Fructoselysine; Receptor; Signal transduction; MonoMac 6

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BBA - General Subjects (v.1760, #11)