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BBA - General Subjects (v.1770, #8)

Editorial Board (pp. ii).

Self-complementary sequences induce the formation of double-stranded filamentous phages by Y. Prieto; O. Sánchez (pp. 1081-1084).

The single strand nature of the filamentous phage (Ff) genome is currently one of the main drawbacks for their application as gene delivery vectors. In this work, by the incorporation of inverted self complementary sequences into the genome of Ff, we were able to convert single strand genome of Ff into double strand DNA structures. The presence of self complementary sequences in phage genome did not affect viral yields significantly, and the formation of double strands structures was successfully determined by a Mung Bean Nuclease resistance assay. Upon transfection into HEK293 cells, the double strand DNA structures showed to be readable by the transcriptional machinery of mammalian cells.

Keywords: Phagemid; Filamentous bacteriophage; Gene transfer; Doubled-stranded

Array-based functional screening for genes that regulate vascular endothelial differentiation of Flk1-positive progenitors derived from embryonic stem cells by Fumio Yamauchi; Mitsuhiro Okada; Koichi Kato; Lars Martin Jakt; Hiroo Iwata (pp. 1085-1097).

Functional genomics is a central topic of current biological research, where a reverse genetic approach is one of the most promising strategies to discover functions of novel genes. Such an approach requires high-throughput methodologies to assess biological functions for a huge number of genes. We have developed a transfection array that permits parallel introduction of multiple plasmids separately into living cells. The feasibility of this array was examined in an assay system. Eleven genes were over-expressed alone, or in combination in vascular progenitors derived from embryonic stem cells. Endothelial differentiation of the cells was monitored through a stably transformed EGFP reporter construct that is expressed only in endothelial cells. Transcriptional activators that promote endothelial differentiation, such as Ets1 and Sox7, were identified. In addition, the assays also revealed an inhibitory effect on endothelial differentiation by several of the factors. These results demonstrate the feasibility of the transfection array for use in cell-based, high-throughput functional assays.

Keywords: Transfection array; Embryonic stem cell; High-throughput screening; Gene function; Endothelial cell; Differentiation

Importance of a hypervariable active-site residue in human Mu class glutathione transferases catalyzing the bioactivation of chemotherapeutic thiopurine prodrugs by Birgitta I. Eklund; Bengt Mannervik (pp. 1098-1103).

Glutathione transferases (GSTs) catalyze the bioactivation of the thiopurine prodrugs azathioprine, cis-6-(2-acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG), thereby releasing the antimetabolites 6-mercaptopurine and 6-thioguanine. In the GST Mu class, GST M1-1 has the highest catalytic efficiency, whereas GST M2-2 and other enzymes are less active. In the evolution of Mu class GSTs, residue 210 appears hypervariable and has particular functional significance. We demonstrate that the catalytic activity of GST M1-1 with cAVTP or tAVTG is successively diminished when wild-type Ser-210 is mutated into Ala followed by Thr. Conversely, mutating wild-type Thr-210 in GST M2-2 into Ala and Ser enhanced the corresponding activities. Comparisons were also made with GST M2-2 distinguished by Gly or Pro in position 210, as well as wild-type GSTs M4-4 and M5-5. The results suggest that the hydroxyl group of Ser in position 210 stabilizes the transition state of the GST-catalyzed reaction. The low activity of GSTs containing Thr in position 210 is probably due to steric hindrance caused by the β-methyl group of the side chain. The ratios of the different catalytic efficiencies were translated into differences in the Gibbs free energies of transition state stabilization. The effects of the mutations were qualitatively parallel for the alternative substrates, but vary significantly in magnitude. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.

Keywords: Glutathione transferase; Thiopurine prodrugs; Functional plasticity; Active-site mutation; Modulated activity

Spermidine triggering effect to the signal transduction through the AtoS–AtoC/Az two-component system in Escherichia coli by Marina C. Theodorou; Evaggelos C. Theodorou; Christos A. Panagiotidis; Dimitrios A. Kyriakidis (pp. 1104-1114).

Recent analysis revealed that, in Escherichia coli the AtoS–AtoC/Az two-component system (TCS) and its target atoDAEB operon regulate the biosynthesis of short-chain poly-( R)-3-hydroxybutyrate (cPHB) biosynthesis, a biopolymer with many physiological roles, upon acetoacetate-mediated induction. We report here that spermidine further enhanced this effect, in E. coli that overproduces both components of the AtoS–AtoC/Az TCS, without altering their protein levels. However, bacteria that overproduce either AtoS or AtoC did not display this phenotype. The extrachromosomal introduction of AtoS–AtoC/Az in an E. coli Δ atoSC strain restored cPHB biosynthesis to the level of the atoSC⁺ cells, in the presence of the polyamine. Lack of enhanced cPHB production was observed in cells overproducing the TCS that did not have the atoDAEB operon. Spermidine attained the cPHB enhancement through the AtoC/Az response regulator phosphorylation, since atoC phosphorylation site mutants, which overproduce AtoS, accumulated less amounts of cPHB, compared to their wild-type counterparts. Exogenous addition of N⁸-acetyl-spermidine resulted in elevated amounts of cPHB but at lower levels than those attained upon spermidine addition. Furthermore, AtoS–AtoC/Az altered the intracellular distribution of cPHB according to the inducer recognized by the TCS. Overall, AtoS–AtoC/Az TCS was induced by spermidine to regulate both the biosynthesis and the intracellular distribution of cPHB in E. coli.

Keywords: Abbreviations; Az; antizyme; ODC; ornithine decarboxylase; TCS; two-component system; HK; histidine kinase; RR; response regulator; SCFA; short-chain fatty acid; PHAs; polyhydroxyalkanoates; PHB; poly-(; R; )-3-hydroxybutyrate; cPHB; complexed poly-(; R; )-3-hydroxybutyrate; atoSC; ⁺; genetic locus encoding the AtoS, AtoC proteinsAntizyme; atoDAEB; AtoS; AtoC; Two-component system; Polyamines; Spermidine; poly-(; R; )-3-hydroxybutyrate

Dynamics of actin filaments during tension-dependent formation of actin bundles by Hiroaki Hirata; Hitoshi Tatsumi; Masahiro Sokabe (pp. 1115-1127).

The actin cytoskeleton stress fiber is an actomyosin-based contractile structure seen as a bundle of actin filaments. Although tension development in a cell is believed to regulate stress fiber formation, little is known for the underlying biophysical mechanisms. To address this question, we examined the effects of tension on the behaviors of individual actin filaments during stress fiber (actin bundle) formation using cytosol-free semi-intact fibroblast cells that were pre-treated with the Rho kinase inhibitor Y-27632 to disassemble stress fibers into a meshwork of actin filaments. These filaments were sparsely labeled with quantum dots for live tracking of their motions. When ATP and Ca²⁺ were applied to the semi-intact cells to generate actomyosin-based forces, actin meshwork in the protruded lamellae was dragged toward the cell body, while the periphery of the meshwork remained in the original region, indicating that centripetally directed tension developed in the meshwork. Then the individual actin filaments in the meshwork moved towards the cell body accompanied with sudden changes in the direction of their movements, finally forming actin bundles along the direction of tension. Dragging the meshwork by externally applied mechanical forces also exerted essentially the same effects. These results suggest the existence of tension-dependent remodeling of cross-links within the meshwork during the rearrangement of actin filaments, thus demonstrating that tension is a key player to regulate the dynamics of individual actin filaments that leads to actin bundle formation.

Keywords: Stress fiber; Semi-intact cell; Self-organization; Actomyosin interaction; Tension; Quantum dot

Extra-pancreatic insulin production in RAt lachrymal gland after streptozotocin-induced islet β-cells destruction by Daniel Andrade Cunha; Mônica C. de Alves; Luiz Fabrizio Stoppiglia; Angélica Gobbi Jorge; Carolina Maria Módulo; Everardo M. Carneiro; Antonio C. Boschero; Mário J.A. Saad; Lício A. Velloso; Eduardo M. Rocha (pp. 1128-1135).

Previous work has revealed that insulin is secreted in the tear film; its mRNA is expressed in the lachrymal gland (LG) and its receptor in tissues of the ocular surface. To test the hypothesis of insulin production in the LG, we compared normal and diabetic rats for: (1) the presence of insulin and C-peptide, (2) glucose- and carbachol-induced insulin secretion ex-vivo, and (3) biochemical and histological characteristics of diabetic LG that would support this possibility. Four weeks after streptozotocin injection, blood and tears were collected from streptozotocin-diabetic male Wistar rats. Insulin levels in the tear film rose after glucose stimulation in diabetic rats, but remained unchanged in the blood. Ex vivo static secretion assays demonstrated that higher glucose and 200 μM carbachol significantly increased mean insulin levels from LG samples of both groups. Insulin and C-peptide were expressed in LG of diabetic rats as determined by RIA. Comparable synaptophysin immune staining and peroxidase activity in the LG of both groups suggest that the structure and function of these tissues were maintained. These findings provide evidence of insulin production by LG. Higher expression of reactive oxygen species scavengers may prevent oxidative damage to LG compared to pancreatic beta-cells.

Keywords: Lachrymal gland; Diabetes mellitus; Extra-pancreatic; Tears; Dry eye; Hormone peptides; Insulin; Reactive oxygen species

Mouse recombinant leptin protects human hepatoma HepG2 against apoptosis, TNF-α response and oxidative stress induced by the hepatotoxin–ethanol by Vairappan Balasubramaniyan; Ruchi Shukla; Gopal Murugaiyan; Ramchandra Ramesh Bhonde; Namasivayam Nalini (pp. 1136-1144).

Obesity is a risk factor for hepatocellular carcinoma (HCC) complicated with alcoholic liver disease (ALD) and cryptogenic cirrhosis. Leptin is a 16-kDa antiobesity hormone secreted mainly by adipocytes. The role of leptin on alcohol-mediated effects in cell line is yet to be unraveled. Therefore, we investigated the effect of leptin against ethanol-elicited cytoxicity in human hepatoma cell lines (HepG2). HepG2 cells were treated with leptin (31.2 nM), ethanol (500 mM), ethanol+leptin and untreated cells served as control. 48 h after treatment, cell viability, apoptosis, TNF-α secretory response and oxidative damage were analysed. Our results suggest that leptin at a concentration of 31.2 nM prevents ethanol elicited cytotoxicity as evidenced by MTT and trypan blue dye exclusion assay. Leptin also inhibited ethanol-induced apoptosis, which was confirmed by [³H] thymidine uptake and cell cycle analysis using propidium iodide (PI) staining. Further, simultaneous leptin treatment along with ethanol showed protection against ethanol mediated cellular damage as indicated by significantly decreased levels of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) and significantly increased levels of reactive nitrogen species (RNS), reduced glutathione (GSH) and elevated activities of superoxide dismutase (SOD) and catalase (CAT). In addition, leptin downregulated the secretion of tumor necrosis factor-α (TNF-α) by ethanol-induced HepG2 cells. Our results demonstrate that simultaneous leptin treatment along with ethanol could be useful in preventing the damage produced by ethanol, which might be of therapeutic interest.

Keywords: Alcoholic liver disease; Antiobesity hormone-leptin; Hepatocellular carcinoma cell lines; Tumor necrosis factor; Reactive oxygen species

Interaction of receptor-activity-modifying protein1 with tubulin by Thomas H. Kunz; Sarah Mueller-Steiner; Kerstin Schwerdtfeger; Peter Kleinert; Heinz Troxler; Jens M. Kelm; Lars M. Ittner; Jan A. Fischer; Walter Born (pp. 1145-1150).

Receptor-activity-modifying protein (RAMP) 1 is an accessory protein of the G protein-coupled calcitonin receptor-like receptor (CLR). The CLR/RAMP1 heterodimer defines a receptor for the potent vasodilatory calcitonin gene-related peptide. A wider tissue distribution of RAMP1, as compared to that of the CLR, is consistent with additional biological functions. Here, glutathione S-transferase (GST) pull-down, coimmunoprecipitation and yeast two-hybrid experiments identified β-tubulin as a novel RAMP1-interacting protein. GST pull-down experiments indicated interactions between the N- and C-terminal domains of RAMP1 and β-tubulin. Yeast two-hybrid experiments confirmed the interaction between the N-terminal region of RAMP1 and β-tubulin. Interestingly, α-tubulin was co-extracted with β-tubulin in pull-down experiments and immunoprecipitation of RAMP1 coprecipitated α- and β-tubulin. Confocal microscopy indicated colocalization of RAMP1 and tubulin predominantly in axon-like processes of neuronal differentiated human SH-SY5Y neuroblastoma cells. In conclusion, the findings point to biological roles of RAMP1 beyond its established interaction with G protein-coupled receptors.

Keywords: Abbreviations; AM; adrenomedullin; CGRP; calcitonin gene-related peptide; CLR; calcitonin receptor-like receptor; CTR; calcitonin receptor; DMEM; Dulbecco's modified Eagle medium; DTT; dithiothreitol; GST; glutathione S-transferase; MALDI-TOF-MS; matrix-assisted laser desorption ionization time-of-flight mass spectrometry; PAGE; polyacrylamide gel electrophoresis; RAMP; receptor-activity-modifying protein; SDS; sodium dodecyl sulphateCalcitonin gene-related peptide; Calcitonin receptor-like receptor; β-tubulin; Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS); Protein interaction; Receptor-activity-modifying protein

Mutations of Trp275 and Trp397 altered the binding selectivity of Vibrio carchariae chitinase A by Wipa Suginta; Chomphunuch Songsiriritthigul; Archara Kobdaj; Rodjana Opassiri; Jisnuson Svasti (pp. 1151-1160).

Point mutations of the active-site residues Trp168, Tyr171, Trp275, Trp397, Trp570 and Asp392 were introduced to Vibrio carchariae chitinase A. The modeled 3D structure of the enzyme illustrated that these residues fully occupied the substrate binding cleft and it was found that their mutation greatly reduced the hydrolyzing activity against pNP-[GlcNAc]₂ and colloidal chitin. Mutant W397F was the only exception, as it instead enhanced the hydrolysis of the pNP substrate to 142% and gave no activity loss towards colloidal chitin. The kinetic study with the pNP substrate demonstrated that the mutations caused impaired K_m and k_cat values of the enzyme. A chitin binding assay showed that mutations of the aromatic residues did not change the binding equilibrium. Product analysis by thin layer chromatography showed higher efficiency of W275G and W397F in G4–G6 hydrolysis over the wild type enzyme. Though the time course of colloidal chitin hydrolysis displayed no difference in the cleavage behavior of the chitinase variants, the time course of G6 hydrolysis exhibited distinct hydrolytic patterns between wild-type and mutants W275G and W397F. Wild type initially hydrolyzed G6 to G4 and G2, and finally G2 was formed as the major end product. W275G primarily created G2–G5 intermediates, and later G2 and G3 were formed as stable products. In contrast, W397F initially produced G1–G5, and then the high- M_r intermediates (G3–G5) were broken down to G1 and G2 end products. This modification of the cleavage patterns of chitooligomers suggested that residues Trp275 and Trp397 are involved in defining the binding selectivity of the enzyme to soluble substrates.

Keywords: Abbreviations; Gn; β; -1–4 linked oligomers of GlcNAc residues where; n; =; 1–6; p; NP-(GlcNAc); ₂; 4-nitrophenyl; N,N′; -diacetyl-; β; -; d; -chitobioside; TLC; thin-layer chromatography; DMAB; p-; dimethylaminobenzaldehyde; IPTG; isopropyl thio-; β; -; d; -galactoside; PMSF; phenylmethylsulphonylfluorideActive-site mutation; Chitin hydrolysis; Chitinase A; Chitooligosaccharide; Specific hydrolyzing activity; Thin layer chromatography; Vibrio carchariae

Four disulfide-bridged scorpion beta neurotoxin CssII: Heterologous expression and proper folding in vitro by Georgina Estrada; Blanca I. Garcia; Emanuele Schiavon; Ernesto Ortiz; Sandrine Cestele; Enzo Wanke; Lourival D. Possani; Gerardo Corzo (pp. 1161-1168).

The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and a FXa proteolytic cleavage region. This recombinant vector was transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The level of expression was 24.6 mg/l of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7538.6 Da as expected. Since native CssII (nCssII) is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1 Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na_v1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.

Keywords: Centruroides suffusus suffusus; Expression; Protein folding; Recombinant; Scorpion; toxin

Trichosanthin induced apoptosis in HL-60 cells via mitochondrial and endoplasmic reticulum stress signaling pathways by Jie Li; Xuechun Xia; Yibao Ke; Huiling Nie; Mark A. Smith; Xiongwei Zhu (pp. 1169-1180).

Trichosanthin (TCS), a traditional Chinese medicine, exerts antitumor activities by inducing apoptosis in many different tumor cell lines. However, the mechanisms remain obscure. The present study focused on various caspase pathways that may be involved in TCS-induced apoptosis in leukemia HL-60 cells. Key caspases in both intrinsic and extrinsic pathways including caspase-8, -9 and -3 were activated upon TCS treatment. Additionally, TCS treatment induced upregulation of BiP and CHOP and also activated caspase-4, which for the first time strongly supported the involvement of endoplasmic reticulum stress pathway in TCS-induced apoptosis. Interestingly, although caspase-8 was activated, Fas/Fas ligand pathway was not involved as evidenced by a lack of induction of Fas or Fas ligand and a lack of inhibitory effect of anti-Fas blocking antibody on TCS-induced apoptosis. Instead, caspase-8 was activated in a caspase-9 and -4 dependent manner. The involvement of mitochondria was demonstrated by the reduction of mitochondrial membrane potential and release of cytochrome c and Smac besides the activation of caspase-9. Further investigation confirmed that caspase-3 was the major executioner caspase downstream to caspase-9, -4 and -8. Taken together, our results suggested that TCS-induced apoptosis in HL-60 cells was mainly mediated by mitochondrial and ER stress signaling pathways via caspase-3.

Keywords: Abbreviations; TCS; trichosanthin; RIP; ribosome-inactivating protein; ER; endoplasmic reticulum; DEVD-CHO; N; -acetyl–Asp–Glu–Val–Asp aldehyde; z-VAD-FMK; benzyloxycarbonyl–Val–Ala–Asp–fluoromethylketone; z-IETD-FMK; Z–Ile–Glu–Thr–Asp–fluoromethylketone; z-LEHD-FMK; z–Leu–Glu–(O–ME)–His–Asp(O–Me) fluoromethyl ketone; cyto c; cytochrome; c; z-YVAD-FMK; Z–Tyr–Val–Ala–Asp–(OMe)–fluoromethylketone; COX IV; Cytochrome C Oxidase subunit IV, Ac, acetateTrichosanthin; Apoptosis; Caspase; Mitochondria; Endoplasmic reticulum stress

A cell model system to study regulation of phosphotidylinositol 3-kinase and protein kinase B activity by cytokines/growth factors produced by type I collagen stimulated immune cells from patients with systemic sclerosis by Thomas M. Chiang; Arnold E. Postlethwaite (pp. 1181-1186).

We have reported that posttranslational modification of systemic sclerosis patients' platelet phosphoinositide 1,3,4,5 kinase (PI 3-K) and protein kinase B (Akt) altered their enzymatic activities. In the present investigation, we have established a cell line model to study further the effects of posttranslational modification and modification by cytokines or growth factors of these two enzymes. Results from these studies suggest that posttranslational modification by phosphorylation of Akt and nitrotyrosylation of PI 3-K increases enzymatic activities, as was observed from SSc patients' platelets. These two signaling components are controlled by a different mechanism, which alters platelet reactivity towards the matrix components of vascular walls. We have used a megakaryotic cell line to study these two enzymes in the presence of cultured supernatants from peripheral blood mononuclear cells (PBMC), which were isolated from blood of SSc patients compared to controls including culture medium, rheumatoid arthritis, systemic lupus erythematosus, and osteoarthritis. The effect of the supernatants from SSc CI-stimulated PBMC cultures on both PI 3-K and Akt is specific.

Keywords: Systemic sclerosis; platelet; nitrotyrosylation of protein; nitric oxide synthase; collagen; protein kinase B

Sarco(endo)plasmic reticulum calcium pump isoforms in paralyzed rat slow muscle by Robert J. Talmadge; Michael Paalani (pp. 1187-1193).

To assess the influence of paralysis on the expression of phenotypic protein isoforms related to muscle relaxation, the effects of spinal cord transection (ST) on sarco(endo)plasmic reticulum calcium ATPase (SERCA) pump isoform protein levels in the slow rat soleus were measured. Western blotting using SERCA isoform specific antibodies demonstrated a rapid up-regulation (7 days post ST) of the fast fiber type-specific isoform (SERCA1). In contrast, the slow fiber type-specific isoform, SERCA2, was decreased with a slower time-course. The up-regulation of SERCA1 protein preceded the up-regulation of fast myosin heavy chain (MyHC) (i.e., MyHC-II). Immunohistochemical analyses of single muscle fibers showed that 15 days after ST there was a pronounced increase in the proportion of slow MyHC fibers with SERCA1 confirming that SERCA1 was up-regulated in the slow fibers of the soleus prior to MyHC-II. These data suggest that the expression of the SERCA isoforms (particularly SERCA1) may serve as more sensitive markers of phenotypic adaptation in response to altered levels of contractile activity than the MyHC isoforms. In addition, since the expression of SERCA isoforms was dissociated from MyHC isoforms, regulation of gene expression for these two different protein systems must involve different signaling events and/or synthetic processes.

Keywords: Myosin heavy chain; Paralysis; Sarco(endo)plasmic reticulum Ca; ²⁺; -ATPase; Skeletal muscle; Spinal cord injury

Lysophosphatidic acid prevents apoptosis of Caco-2 colon cancer cells via activation of mitogen-activated protein kinase and phosphorylation of Bad by Raluca Rusovici; Amr Ghaleb; Hyunsuk Shim; Vincent W. Yang; C. Chris Yun (pp. 1194-1203).

Lysophosphatidic acids (LPA) exert growth factor-like effects through specific G protein-coupled receptors. The presence of different LPA receptors often determines the specific signaling mechanisms and the physiological consequences of LPA in different environments. Among the four members of the LPA receptor family, LPA₂ has been shown to be overexpressed in colon cancer suggesting that the signaling by LPA₂ may potentiate growth and survival of tumor cells. In this study, we examined the effect of LPA on survival of colon cancer cells using Caco-2 cells as a cell model system. LPA rescued Caco-2 cells from apoptosis elicited by the chemotherapeutic drug, etoposide. This protection was accompanied by abrogation of etoposide-induced stimulation of caspase activity via a mechanism dependent on Erk and PI3K. In contrast, perturbation of cellular signaling mediated by the LPA₂ receptor by knockdown of a scaffold protein NHERF2 abrogated the protective effect of LPA. Etoposide decreased the expression of Bcl-2, which was reversed by LPA. Etoposide decreased the phosphorylation level of the proapoptotic protein Bad in an Erk-dependent manner, without changing Bad expression. We further show that LPA treatment resulted in delayed activation of Erk. These results indicate that LPA protects Caco-2 cells from apoptotic insult by a mechanism involving Erk, Bad, and Bcl-2.

Keywords: Lysophosphatidic acids; MAPK; Receptor; Apoptosis

Sugar-dependent photodynamic effect of glycoconjugated porphyrins: A study on photocytotoxicity, photophysical properties and binding behavior to bovine serum albumin (BSA) by Makoto Obata; Shiho Hirohara; Kohei Sharyo; Hiroki Alitomo; Kazumi Kajiwara; Shin-ichi Ogata; Masao Tanihara; Chikara Ohtsuki; Shigenobu Yano (pp. 1204-1211).

The photocytotoxicity of four glycoconjugated porphyrins, namely 5,10,15,20-tetrakis[4-(β-d-glucopyranosyloxy)phenyl]porphyrin ( p-1a), 5,10,15,20-tetrakis[4-(β-d-galactopyranosyloxy)phenyl]porphyrin ( p-1b), 5,10,15,20-tetrakis[4-(β-d-xylopyranosyloxy)phenyl]porphyrin ( p-1c) and 5,10,15,20-tetrakis[4-(β-d-arabinopyranosyloxy)phenyl]porphyrin ( p-1d), was evaluated in HeLa cells in the concentration range from 1 to 7 μM using a light dose of 16 J·cm−² with a wavelength greater than 500 nm. The photocytotoxicity depends on the sugar moieties, and increases in the order of p-1d< p-1a< p-1b< p-1c. The order of the photocytotoxicity is at variance with that of the cellular uptake reported previously. On the other hand, the photophysical properties of the glycoconjugated porphyrins also depends on the sugar moieties in physiological media such as phosphate buffered saline (PBS) containing 10 wt.% bovine serum albumin (BSA). In particular, the oscillator strength in the range above 500 nm increases in the order of p-1d= p-1a< p-1c< p-1b, which is good agreement with the order of the photocytotoxicity in HeLa cells. The interaction between the glycoconjugated porphyrins and BSA was evaluated by means of electronic absorption, fluorometric and circular dichroic (CD) titrations. Fluorometric titration showed no differences in the apparent binding constants, K, between the glycoconjugated porphyrins p-1a, p-1b, p-1c and p-1d. On the other hand, the number of binding sites, n, depends on the sugar moieties of the glycoconjugated porphyrin, and increases in the order of p-1b< p-1a< p-1d< p-1c. CD titration was also characterized by the n value determined by fluorometric titration, suggesting the n value is a good descriptor for the interaction between glycoconjugated porphyrins and BSA. However, it was found that the n value was poorly related to the photophysical properties in physiological media and the photocytotoxicity. Even though the role of the sugar moieties on the photodynamic effect is not fully understood, the photophysical properties of the glycoconjugated porphyrins are strongly modulated by the physiological media resulting in the sugar-dependent photocytotoxicity.

Keywords: Glycoconjugated porphyrins; Bovine serum albumin (BSA); Cellular uptake; Fluorometric titration; Circular dichroic (CD) titration; Photodynamic therapy (PDT)

Reduction of vanadium(V) to vanadium(IV) by NADPH, and vanadium(IV) to vanadium(III) by cysteine methyl ester in the presence of biologically relevant ligands by Mohammad K. Islam; Chieko Tsuboya; Hiroko Kusaka; Sen-ichi Aizawa; Tatsuya Ueki; Hitoshi Michibata; Kan Kanamori (pp. 1212-1218).

To better understand the mechanism of vanadium reduction in ascidians, we examined the reduction of vanadium(V) to vanadium(IV) by NADPH and the reduction of vanadium(IV) to vanadium(III) byl-cysteine methyl ester (CysME). UV-vis and electron paramagnetic resonance spectroscopic studies indicated that in the presence of several biologically relevant ligands vanadium(V) and vanadium(IV) were reduced by NADPH and CysME, respectively. Specifically, NADPH directly reduced vanadium(V) to vanadium(IV) with the assistance of ligands that have a formation constant with vanadium(IV) of greater than 7. Also, glycylhistidine and glycylaspartic acid were found to assist the reduction of vanadium(IV) to vanadium(III) by CysME.

Keywords: Vanadium; Ascidian; NADPH; Biologically relevant ligands; Reduction

Biochemical characterization of a N-terminal fragment (p5) cleaved from fibroblast growth factor-binding protein (FGF-BP) in bovine milk in vitro by Kenzo Ohtsuki; Kyoko Hirayama; Fumitaka Kawakami; Tomoki Kato; Hiroshi Kawakami (pp. 1219-1229).

By means of successive gel filtration on a Superdex 30 pg column and Mono S column chromatography, a 5-kDa polypeptide (p5) was highly purified from the low molecular weight (LMW) fraction separated from the partially purified lactoferrin (bLF) fraction of bovine milk, and biochemically characterized as a phosphate acceptor for two protein kinases [cAMP-dependent protein kinase (PKA) and casein kinase 1δ (CK1δ)] in vitro. Purified p5 was identified as a fragment (N-terminal positions 24–51, 28 amino acid residues) cleaved from fibroblast growth factor-binding protein (FGF-BP, p37). Both purified p5 and synthetic p5 (sp5) were effectively phosphorylated by PKA, and also phosphorylated by CK1δ in the presence of two sulfated lipids [sulfatide or cholesterol-3-sulfate (CH-3S), SCS] in vitro. A novel phosphorylation site (RNRRGS) for CK1δ and a potent SCS-binding site (RNRR) on p5 were identified. The PKA-mediated phosphorylation of p5 was highly stimulated when incubated with either acidic FGF (aFGF) or bLF in vitro, but this phosphorylation was more sensitive to SCS than H-89 (a specific PKA inhibitor). Immunoprecipitate experiments revealed p5, but not the phosphorylated p5, to be directly bound to aFGF in vitro. These results show that (i) p5 has a high binding affinity with aFGF as well as bLF; (ii) the binding of SCS to p5 results in the selective inhibition of its phosphorylation by PKA; and (iii) SCS functions as an effective stimulator for the phosphorylation of p5 by CK1δ in vitro. In addition, p5 may play an important physiological role as a trafficking factor for the physiological interaction between aFGF group including endothelial cell growth factors and their binding proteins in vivo.

Keywords: Abbreviations; CH-3S; cholesterol-3-sulfate; CK1δ; casein kinase 1δ; CK2; casein kinase 2; DTT; dithiothreitol; FGF; fibroblast growth factor; aFGF; acidic FGF; bFGF; basic FGF; FGF-BP; FGF-binding protein; FGFR; FGF receptor; bLF; bovine lactoferrin; bLF-BP; bLF-binding protein; LMW fraction; low molecular weight fraction; p5; 5 kDa polypeptide; PKA; cAMP-dependent protein kinase; C-kinase; Ca; ²⁺; - and phospholipid-dependent protein kinase; PMSF; phenylmethyl sulfonyl fluoride; SCS; sulfatide and CH-3S; SCS-BP; SCS-binding protein; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; sp5; synthetic p5FGF-binding protein; N-terminal fragment of FGF-BP; cAMP-dependent protein kinase; Casein kinase 1δ; Phosphorylation; Sulfatide; Cholesterol-3-sulfate; Bovine milk

Nitric oxide induces apoptosis in cutaneous T cell lymphoma (HuT-78) by downregulating constitutive NF-κB by Loveena Rishi; Rohan Dhiman; Manoj Raje; Sekhar Majumdar (pp. 1230-1239).

Constitutive active NF-κB have been shown to protect cutaneous T cell lymphoma (CTCL) cells from apoptosis. In the present study, we have studied the cytotoxic potential of nitric oxide generating compound, sodium nitroprusside (SNP) on CTCL cell line, HuT-78. Treatment of cells with SNP resulted in decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and poly (ADP ribose) polymerase cleavage. SNP treatment inhibited activation of NF-κB in a concentration dependent manner. SNP increased the expression of IκBα without affecting the phosphorylation of IκBα. Downregulation of NF-κB by SNP decreased p65 nuclear translocation as evident by confocal fluorescence microscopy. Further it was found that SNP treatment caused downregulation of Bcl-2 family member (Bcl-xl) in HuT-78 cells. Thus, we have provided evidence that SNP induces apoptosis in CTCL cell line, HuT-78 by downregulating constitutive NF-κB and thereby Bcl-xl expression.

Keywords: Cutaneous T cell lymphoma; Nitric oxide; Apoptosis

Detection and biochemical characterisation of a novel polymorphism in the human GSTP1 gene by Neil R. Kitteringham; Luke Palmer; Andrew Owen; Lu-Yun Lian; Roz Jenkins; Sam Dowdall; Ian Gilmore; B. Kevin Park; Chris E. Goldring (pp. 1240-1247).

The glutathione transferases (GSTs) mediate the detoxification of a broad spectrum of electrophilic chemicals. We report here the identification and characterisation of a novel naturally occurring transition that changes codon 169 from GGC (Gly) to GAC (Asp) in the human Pi class GST, GSTP1. Expression of the variant in human HepG2 cells led to a small increase in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation compared to the wild-type protein. Asp¹⁶⁹ GSTP1-1 expressed at high levels in Escherichia coli displayed a small but significant increase in specific activity towards CDNB compared to Gly¹⁶⁹ GSTP1-1. The catalytic efficiency with CDNB was higher for Asp¹⁶⁹ GSTP1-1 compared to the wild-type enzyme, although the kinetic constants of the mutant and the wild-type enzyme towards glutathione were not different. Modelling indicated that the mutation does not appear to change protein conformation. The distribution of the genotypes in a normal healthy population (217 individuals) was 94.3% for the Gly/Gly genotype and 5.7% for the Gly/Asp genotype; no Asp/Asp genotypes were detected in this population. The frequency of the Asp¹⁶⁹ allele in the only oxidative stress-linked pathology that we have studied to date, i.e. alcoholic liver disease, was not significantly different from healthy controls. In conclusion, we have detected and characterised a novel SNP in GSTP1 that may play a role in modulating the activity of GSTP1-1.

Keywords: Abbreviations; GST; glutathione transferase; PCR; polymerase chain reaction; CDNB; 1-chloro-2,4-dinitrobenzene; BITC; benzylisothiocyanate; EA; ethacrynic acid; ALD; alcoholic liver disease; ROS; reactive oxygen species; MALDI; matrix assisted laser desorption ionization; SNP; single nucleotide polymorphism; rmsd; relative mean square deviation; CD; circular dichroismGlutathione transferase P1; Polymorphism; Alcoholic liver disease; Genotype

Molecular modeling and functional analysis of the AtoS–AtoC two-component signal transduction system of Escherichia coli by A.I. Grigoroudis; C.A. Panagiotidis; E.E. Lioliou; M. Vlassi; D.A. Kyriakidis (pp. 1248-1258).

The AtoS–AtoC two-component signal transduction system positively regulates the expression of the atoDAEB operon in Escherichia coli. Upon acetoacetate induction, AtoS sensor kinase autophosphorylates and subsequently phosphorylates, thereby activating, the response regulator AtoC. In a previous work we have shown that AtoC is phosphorylated at both aspartate 55 and histidine73. In this study, based on known three-dimensional structures of other two component regulatory systems, we modeled the 3D-structure of the receiver domain of AtoC in complex with the putative dimerization/autophosphorylation domain of the AtoS sensor kinase. The produced structural model indicated that aspartate 55, but not histidine 73, of AtoC is in close proximity to the conserved, putative phosphate-donor, histidine (H398) of AtoS suggesting that aspartate 55 may be directly involved in the AtoS–AtoC phosphate transfer. Subsequent biochemical studies with purified recombinant proteins showed that AtoC mutants with alterations of aspartate 55, but not histidine 73, were unable to participate in the AtoS–AtoC phosphate transfer in support of the modeling prediction. In addition, these AtoC mutants displayed reduced DNA-dependent ATPase activity, although their ability to bind their target DNA sequences in a sequence-specific manner was found to be unaltered.

Keywords: Abbreviations; TCS; two-component system; HK; histidine kinase; RR; response regulator; trAtoC; truncated form of AtoC; IPTG; isopropyl-; β; -; d; -thiogalactoside; PMSF; phenylmethylsulfonylfluorideAtoS; AtoC; Two-component system; Antizyme; ATPase; DNA-binding; σ; ⁵⁴; -regulator factor; Structural model

NTPDase and 5′ ecto-nucleotidase expression profiles and the pattern of extracellular ATP metabolism in the Walker 256 tumor by A. Buffon; M.R. Wink; B.V. Ribeiro; E.A. Casali; T.A. Libermann; L.F. Zerbini; S.C. Robson; J.J.F. Sarkis (pp. 1259-1265).

In this study, we evaluated the NTPDases and ecto-5′-nucleotidase (CD73) expression profiles and the pattern of adenine nucleotide hydrolysis in rats submitted to the Walker 256 tumor model, 6, 10 and 15 days after the subcutaneous inoculation. Using RT-PCR analysis, we identified mRNA for all of the members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated and a 5′-nucleotidase. By quantitative real-time PCR, Entpd1 ( Cd39) and Entpd2 (Cd39L1) and CD73 were identified as the dominant genes expressed by the Walker 256 tumor, at all times studied. Extracellular adenine nucleotide hydrolysis by the Walker 256 tumor was estimated by HPLC analysis. Rapid hydrolysis of extracellular ATP by the tumor cells was observed, leading to the formation of adenosine and inosine in cells obtained from solid tumors at 6 and 10 days after inoculation. Cells obtained from solid tumors at 15 days of growth presented high levels of AMP and presented adenosine as a final product after 90 min of incubation. Results demonstrate that the presence of NTPDases and 5′-nucleotidase enzymes in Walker 256 tumor cells may be important for regulation of the extracellular adenine nucleotides/adenine nucleoside ratio, therefore leading to tumor growth.

Keywords: Walker 256 tumor; NTPDases; 5′-nucleotidase; Adenine nucleotides; Cancer

Site-directed mutagenesis of glutamate 317 of bovine α-1,3Galactosyltransferase and its effect on enzyme activity: Implications for reaction mechanism by Patricia Molina; Ronald M.A. Knegtel; Bruce A. Macher (pp. 1266-1273).

Bovine α1,3galactosyltransferase (α1,3GalT) transfers galactose from UDP-α-galactose to terminal β-linked galactosyl residues with retention of configuration of the incoming galactose residue. The epitope synthesized has been shown to be critical for xenotransplantation. According to a proposed double-displacement reaction mechanism, glutamate-317 (E317) is thought to be the catalytic nucleophile. The proposed catalytic role of E317 involves an initial nucleophilic attack with inversion of configuration and formation of a covalent sugar–enzyme intermediate between E317 and galactose from the donor substrate, followed by a second nucleophilic attack performed by the acceptor substrate with a second inversion of configuration. To determine whether E317 of α1,3GalT is critical for enzyme activity, site-directed mutagenesis was used to substitute alanine, aspartic acid, cysteine and histidine for E317. If the proposed reaction mechanism for the α1,3GalT enzyme is correct, E317D and E317H would produce active enzymes since they can act as nucleophiles. The non-conservative mutation E317A and conservative mutation E317C are predicted to produce inactive or very low activity enzymes since the E317A mutant cannot engage in a nucleophilic attack, and the E317C mutant would trap the galactose residue. The results obtained demonstrate that E317D and E317H mutants retained activity, albeit significantly less than the wild-type enzyme. Additionally, both E317A and E317C mutant also retained enzyme activity, suggesting that E317 is not the catalytic nucleophile proposed in the double-displacement mechanism. Therefore, either a different amino acid may act as the catalytic nucleophile or the reaction must proceed by a different mechanism.

Keywords: Abbreviations; α1,3GalT; alpha 1,3-galactosyltransferase; LacNAc-C; ₈; Galβ1,4GlcNAc-O-(CH; ₂; ); ₈; COOCH; ₃; LacNAc-sp-biotin; Galβ1,4GlcNAc-O(CH; ₂; ); ₃; NHCO(CH; ₂; ); ₅; NH-biotin; nLc; ₄; Cer; neolactotetraosylceramide or Galβ1,4GlcNAcβ1,3Galβ1,4Glcβ1,1Cer; PCR; polymerase chain reaction; UDP-Gal; UDP-α-galactose; UDP-Glc; UDP-glucoseSite-directed mutagenesis; α-1,3Galactosyltransferase; Double-displacement reaction mechanism; Modeling

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BBA - General Subjects (v.1770, #8)