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BBA - General Subjects (v.1725, #2)

Editorial Board (pp. ii).

In vitro platination of human breast cancer suppressor gene1 (BRCA1) by the anticancer drug carboplatin by Adisorn Ratanaphan; Bhutorn Canyuk; Siriwat Wasiksiri; Pawika Mahasawat (pp. 145-151).

Carboplatin is an anticancer drug for the treatment of cancers affecting various organs including ovary and testes. It essentially exerts its cytotoxicity against cancerous cells via covalent attachment of platinum atom to DNA, generating various platinum-DNA adducts. Platinum-DNA adducts inhibit biological processes essential for cellular viability. However, carboplatin interacts nonspecifically with DNA, resulting in damaging of normal cell DNA. Potential in vitro interaction of carboplatin with genes encoding tumor suppressor proteins such as human breast cancer suppressor gene 1(BRCA1) was herein investigated. The 696-bp fragment of the 3′-region of BRCA1 gene (nucleotide 4897–5592) was amplified by RT-PCR using mRNA templates isolated from human white blood cells. Retardation of the electrophoretic migration on agarose gel of drug-treated DNA, in the dose–response manner, was observed. Analysis by restriction digestion with PvuII and EcoO109I suggested that the platination favorably occurred at the dGpG sequence of EcoO109I-cleaved site. The semi-quantitative PCR-based assay was used to determine the lesion frequencies produced by carboplatin in the 696-bp fragment of the 3′-region of BRCA1 gene and in the 3,426-bp fragment of the BRCA1 exon 11 of human breast adenocarcinoma MCF-7 cells. A significant decrease in DNA amplification was observed at 400 μM of carboplatin with approximately 1–2 platinum atoms per BRCA1 fragment. Carboplatin caused slightly less damage at equimolar concentrations in cells than in cell-free BRCA1 fragment.

Keywords: BRCA1; Carboplatin; Gel electrophoresis; QPCR

Aggrecanase-1 (ADAMTS-4) interacts with α1-antitrypsin by Koji Yoshida; Yasuyuki Suzuki; Akio Saito; Kanji Fukuda; Chiaki Hamanishi; Hiroshi Munakata (pp. 152-159).

In degradative articular diseases such as rheumatoid arthritis and osteoarthritis, loss of the extracellular matrix occurs, resulting in the destruction of joint cartilage. Proteolysis of aggrecan is one of the early events that leads to breakdown of the extracellular matrix. Aggrecanase-1 (ADAMTS-4) is considered to play a pivotal role in the abrasion of cartilage aggrecan in rheumatoid arthritis and osteoarthritis. To identify an endogenous inhibitor of aggrecanase-1, we performed a yeast two-hybrid screen using the catalytic domain of human aggrecanase-1 as a bait and transformed an EGY48 yeast strain carrying the bait plasmid with a human liver cDNA library plasmid. This screen identified α1-antitrypsin, a member of the family of plasma serine proteinase inhibitors, as a prey. Recombinant aggrecanase-1 and α1-antitrypsin were expressed in mammalian cells and used in coimmunoprecipitation experiments, which showed that full-length aggrecanase-1 and α1-antitrypsin are also associated in vivo. However, aggrecanase-1 did not interfere with the inhibitory activity of α1-antitrypsin against elastase, and α1-antitrypsin had no effect on the proteolytic activity of aggrecanase-1. Taken together, these data suggest that aggrecanase-1 and α1-antitrypsin bind in vivo, although the physiological significance of the interaction between aggrecanase-1 and α1-antitrypsin remains unclear.

Keywords: Aggrecan; Aggrecanase; α1-antitrypsin; Two-hybrid

Purification, characterization, cDNA cloning, and expression of asialofetuin-binding C-type lectin from eggs of shishamo smelt ( Osmerus [ Spirinchus] lanceolatus) by Masahiro Hosono; Shigeki Sugawara; Yukiko Ogawa; Takayuki Kohno; Motoaki Takayanagi; Kazuo Nitta (pp. 160-173).

A novel C-type lectin (OLABL) was isolated from the eggs of shishamo smelt [ Osmerus ( Spirinchus) lanceolatus] by affinity chromatography on asialofetuin-Sepharose. OLABL had a molecular mass of 29 kDa on SDS-PAGE under nonreducing conditions and two subunits with masses of 15 kDa (OLABL-H) and 14 kDa (OLABL-L) under reducing conditions. Thus, OLABL is a heterodimeric protein. cDNA sequence analysis revealed that the H- and L-subunits of OLABL were composed of 137 and 136 amino acid residues, respectively, and showed almost identical (95%) sequences, with slight differences in the N-terminal and C-terminal regions. Since each subunit contained only the characteristic motif of C-type lectin-like domain (CTLD), EPN-E-WND, OLABL is a member of group VII of the CTLD-containing protein family. Although OLABL had an EPN sequence that is known as a mannose-specific motif found in the collectin family, OLABL agglutinated rabbit erythrocytes without the addition of Ca²⁺ ion, and this activity was inhibited byl-rhamnose andd-galactose derivatives, but not byd-mannose andd-glucose. These results indicate that OLABL has similar characteristics to AJL-2, a calcium-independent lactose specific lectin isolated from Japanese eel skin mucus. Recombinant OLABLs (rHisOLABLs), His-tagged homodimers of the H- and L-subunits, were refolded from inclusion bodies expressed by Escherichia coli. rHisOLABL-L was recovered as a soluble form, but rHisOLABL-H was hardly dissolved in a renaturing buffer. The specific activities of rHisOLABL-L, rHisOLABL-H, and native OLABL were 500, 36, and 20, respectively. These findings suggest that the combination of subunits may affect the solubility and activity of these dimeric form lectins.

Keywords: Abbreviations; aa; amino acid; OLABL; asialofetuin-binding lectin from; O; .; lanceolatus; OLRBL; rhamnose-binding lectin from; O; .; lanceolatus; RBL; rhamnose-binding lectin; CRD; carbohydrate recognition domain; PMSF; phenylmethanesulfonyl fluoride; SEF; saline extracted fraction; HU; hemagglutination unit; BSA; bovine serum albumin; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 2-ME; 2-mercaptoethanol; PB; phosphate buffer; PBS; phosphate-buffered saline; TBS; 20 mM Tris–HCl buffer (pH 7.4) containing 0.15 M NaCl; RTase; reverse transcriptase; RACE; rapid amplification of cDNA ends; AUAP; abridged universal amplification primer; GSP; gene-specific primer; LB; Luria-Bertani; IPTG; isopropyl 1-thio-β-; d; -galactoside; Ntr; N-terminal region; Ctr; C-terminal regionC-type lectin; Fish egg; Asialofetuin; Heterodimer; EPN sequence; Calcium-independent

Interdomain interactions regulate the activation of the heme-regulated eIF2α kinase by Bo-Geon Yun; Jessica A.B. Matts; Robert L. Matts (pp. 174-181).

The heme-regulated inhibitor of protein synthesis (HRI) regulates translation through the phosphorylation of the α-subunit of eukaryotic initiation factor-2 (eIF2). While HRI is best known for its activation in response to heme-deficiency, we recently showed that the binding of NO and CO to the N-terminal heme-binding domain (NT-HBD) of HRI activated and suppressed its activity, respectively. Here, we examined the effect of hemin, NO, and CO on the interaction between the NT-HBD and the catalytic domain of HRI (HRI/ΔHBD). Hemin stabilized the interaction of NT-HBD with HRI/ΔHBD, and NO and CO disrupted and stabilized this interaction, respectively. Mutant HRI (ΔH-HRI), lacking amino acids 116–158 from the NT-HBD, was less sensitive to heme-induced inhibition, and mutant NT-HBD lacking these residues did not bind to HRI/ΔHBD. HRI/ΔHBD and ΔH-HRI also activated more readily than HRI in response to heme-deficiency. Thus, HRI's activity is regulated through the modulation of the interaction between its NT-HBD and catalytic domain.

Keywords: Heme-regulated eIF2α kinase; Heme-binding domain; Nitric oxide; Carbon monoxide; Heme; Interdomain interaction

A HER-2/neu peptide admixed with PLA microspheres induces a Th1-biased immune response in mice by Konstantina N. Nikou; Nikolaos Stivaktakis; Konstantinos Avgoustakis; Panagiota A. Sotiropoulou; Sonia A. Perez; Constantin N. Baxevanis; Michael Papamichail; Leondios Leondiadis (pp. 182-189).

The elimination of cancer cells requires strong cellular immune responses, and these responses are induced by the activation of Th1 lymphocytes. In this work, the possibility of inducing a Th1 type of immune response in vivo by mixing a HER-2/neu synthetic CTL (cytotoxic T lymphocyte) peptide [HER-2/neu (789–797)], with poly-lactide (PLA) microspheres was investigated. Various formulations of the peptide were administered to HLA-A2.1 transgenic (HHD) mice. Cellular experiments, assessing proliferation and cytokine determination in splenocyte culture supernatants, were carried out in order to evaluate the type of immune response to the antigen. The in vivo administration of the peptide antigen admixed with the PLA microspheres induced a potent immune response which was comparable to that induced by the combination of the antigen in complete Freund's adjuvant (CFA). Furthermore, the cytokine profile produced by the T lymphocytes of the immunized animals indicated that the combination of the peptide antigen with the PLA microspheres induced a strong Th1 biased immune response to the antigen. The time of peptide incubation with the microspheres prior to administration did not affect the immune response, which further simplifies the preparation of this type of vaccine. The results justify further investigation of the possibility of inducing effective cellular immune responses against cancer cells overexpressing HER-2/neu molecules by simply mixing appropriate HER-2/neu peptide antigens with PLA microspheres.

Keywords: HER-2/neu; Peptide; PLA microsphere; Immune response; Cancer; Vaccine

Characterization of the structure of antithrombin-binding heparan sulfate generated by heparan sulfate 3- O-sulfotransferase 5 by Jinghua Chen; Jian Liu (pp. 190-200).

The 3- O-sulfation of glucosamine is a key modification step during the biosynthesis of anticoagulant heparan sulfate (HS). Both heparan sulfate 3- O-sulfotransferase -1 (3-OST-1) and 3- O-sulfotransferase-5 (3-OST-5) transfer sulfate to the 3-OH group of glucosamine to generate antithrombin-binding heparan sulfate (HS^act). Here, we reported the isolation and characterization of the antithrombin-binding HS oligosaccharides generated by 3-OST-5 (3-OST-5 oligo^act).³H-labeled HS of Chinese hamster ovary cells was exhaustively modified by 3-OST-1 to remove the 3-OST-1 modification sites followed by antithrombin-affinity fractionation. The nonantithrombin-binding fraction of 3-OST-1 pretreated HS was further modified by 3-OST-5 to generate additional antithrombin-binding HS, which was designated as 3-OST-5 HS^act. Structural analysis of 3-OST-5 HS^act revealed that the antithrombin-binding site of 3-OST-5 HS^act is located within a domain clustered with N-sulfated glucosamine units. We also isolated 3-OST-5 antithrombin-binding oligosaccharides (3-OST-5 oligo^act) from high pH nitrous acid degraded 3-OST-5 HS^act. A disaccharide analysis revealed that 3-OST-5 oligo^act were composed of multiple 3- O-sulfated glucosamine units. Our results provide additional insights on the relationship between the anticoagulant activity and structure of HS.

Keywords: Antithrombin; Heparan sulfate; 3-; O; -sulfotransferase-5; Anti-coagulation

Adaptative metabolic response of human colonic epithelial cells to the adverse effects of the luminal compound sulfide by Xavier Leschelle; Marc Goubern; Mireille Andriamihaja; Herv� M. Blotti�re; Elodie Couplan; Maria-del-Mar Gonzalez-Barroso; Caroline Petit; Anthony Pagniez; Catherine Chaumontet; Bernard Mignotte; Fr�d�ric Bouillaud; Fran�ois Blachier (pp. 201-212).

Hydrogen sulfide (H₂S), a bacterial metabolite present in the lumen of the large intestine, is able to exert deleterious effects on the colonic epithelium. The mechanisms involved are still poorly understood, the reported effect of sulfide being its capacity to reduce n-butyrate β-oxidation in colonocytes. In this work, we studied both the acute effect of the sodium salt of H₂S on human colonic epithelial cell metabolism and the adaptative response of these cells to the pre-treatment with this agent. Using the human colon carcinoma epithelial HT-29 Glc^−/+ cell model, we found that the acute effect of millimolar concentrations of NaHS was to inhibitl-glutamine, n-butyrate and acetate oxidation in a dose-dependent manner. Using micromolar concentrations of NaHS, a comparable effect but largely reversible was observed for O₂ consumption and cytochrome c oxidase activity. Pre-treatment with 1 mM NaHS induced several adaptative responses. Firstly, increased lactate release and decreased cellular oxygen consumption evidenced a Pasteur-like effect which only partly compensated for the altered mitochondrial ATP production. Thus, a decrease in the proliferation rate with a constant adenylate charge was observed. Secondly, in these pre-treated cells, NaHS induced a hypoxia-like effect on cytochrome c oxidase subunits I and II which were decreased. Thirdly, a mild uncoupling of mitochondrial respiration possibly resulting from an increase of UCP2 protein was observed. The NaHS antimitotic activity was not due to cellular apoptosis and/or necrosis but to a proportional slowdown in all cell cycle phases. These results are compatible with a metabolic adaptative response of the HT-29 colonic epithelial cells to sulfide-induced O₂ consumption reduction which, through the maintenance of a constant energetic load and an increased mitochondrial proton leak, would participate in the preservation of cellular viability.

Keywords: Abbreviations; AT cells; cells undergoing acute treatment; FCCP; carbonyl cyanide p-trifluoromethoxyphenyl hydrazone; FITC; fluorescein isothiocyanate; LDH; lactate dehydrogenase; PBS; Phosphate Buffer Saline; PFA; paraformaldehyde; PT cells; pre-treated cells; TES; N-tris [hydroxymethyl] methyl-2-aminoethane-sulfonic acid; TPP; ⁺; tetraphenyl phosphonium; TUNEL; terminal deoxyuridine nick end labelling; VADC; voltage dependent anion channelSulfide; Colonic cells; Cytochrome; c; oxidase; UCP2; Cell cycle; Substrate oxidation

Oxidative inactivation of paraoxonase—implications in diabetes mellitus and atherosclerosis by Sonia-Athina P. Karabina; Alexander N. Lehner; Elizabeth Frank; Sampath Parthasarathy; Nalini Santanam (pp. 213-221).

Human serum paraoxonase (PON1) has been implicated to play an important role in cardiovascular disease and diabetes. Studies in the literature indicate that PON1 has two different enzyme activities, i.e., esterase and hydroperoxide reducing activities. The objective of this study was to establish the importance of these two activities and to distinguish between them. As the addition of copper immediately inactivated the enzyme, we used auto-oxidation as the model system. Auto-oxidation of HDL resulted in more than 80% reduction of the esterolytic activity, which was protected by antioxidants, Vitamin E (50%) and PDTC (95%) and completely by 1 M glucose. In contrast, the hydroperoxide reducing activity, using unesterified hydroperoxides remained unaffected with time. We also used pNPHPODE (novel substrate) to establish that hydrolysis might be a prerequisite for the enzyme to act on the esterified hydroperoxide. The results indicated that the hydrolysis of the substrate was inhibited under oxidizing conditions with no reduction of the hydroperoxide. Overall, our findings suggest that protecting the esterolytic activity of PON1 by antioxidants might be important in preserving its action on phospholipid peroxides and a concerted reaction involving the esterolytic and hydroperoxide reducing activities might be suggested for the action of PON1.

Keywords: Lipid hydroperoxide; High density lipoprotein; Antioxidant; Glucose; Esterase; Low density lipoprotein

Evidence for lectin activity of a plant receptor-like protein kinase by application of neoglycoproteins and bioinformatic algorithms by Sabine Andr�; Hans-Christian Siebert; Mitsuru Nishiguchi; Kiyoshi Tazaki; Hans-Joachim Gabius (pp. 222-232).

Detection of genes for putative receptor-like protein kinases, which contain an extracellular domain related to leguminous lectins, in plant genomes inspired the hypothesis that this part acts as sensor. Initial support for this concept came from proof for protein kinase activity. The next step, focusing on the protein of lombardy poplar ( Populus nigra var. italica), is scrutiny for lectin activity. Consequently, we first pinpointed sets of high-scoring sequence pairs by extensive databank search. The calculations resulted in P-values in the range from 10⁻¹⁴ to 10⁻¹⁸ exclusively for leguminous lectins, the Pterocarpus angolensis agglutinin being frontrunner with P=3×10⁻¹⁸ and thus most suitable template for modeling. The superimposition of the two folds gave notable similarity in the region responsible for binding carbohydrate and Ca²⁺/Mn²⁺-ions. Binding activity toward carbohydrates was detected by assaying a panel of (neo)glycoproteins as polyvalent probes, especially for α-l-rhamnose and glycans of asialofetuin. It was strictly dependent on Ca²⁺-ions, enhanced by Mn²⁺-ions and reached a K_D-value of 34.3 nM for the neoglycoprotein with rhamnose as ligand. These results give further research direction to define physiological ligands, plant/bacterial rhamnose-containing saccharides and rhamnose-mimetic glycans or peptides being potential candidates.

Keywords: Agglutinin; Kinase; Lectin; Neoglycoprotein; Rhamnose; Signaling

Immunostimulation effects of proteose-peptone component 3 fragment on human hybridomas and peripheral blood lymphocytes by Takuya Sugahara; Hiroyuki Onda; Yusuke Shinohara; Mayumi Horii; Koichi Akiyama; Katsunori Nakamoto; Kazushi Hara (pp. 233-240).

Fat-free bovine milk fermented by 12 kinds of lactic acid bacteria and yeast enhanced monoclonal antibody production of human hybridoma HB4C5 cells 2.8-fold in serum-free medium. Immunoglobulin production of human peripheral blood lymphocytes (PBL) was also stimulated in vitro. IgM and IgG production of human PBL was accelerated up to 2.8-fold and 5.4-fold, respectively. Interferon-γ production of human PBL was also accelerated 6.0-fold by 50 μg/ml of the fermented milk. However, interleukin-4 production of PBL was not affected, and tumor necrosis factor-α production was suppressed. The activity was enhanced 2.5-fold by the thermal treatment for 30 min at 65 °C and was completely lost by trypsin digestion. The findings suggested that the active substance in the fermented milk was heat stable protein. Gel-filtration and the SDS-PAGE analysis revealed that the molecular weight of the active substance was estimated as 19.0 kDa, which was not detected in fat-free bovine milk before fermentation. N-terminal amino acid sequence of the 19.0 kDa protein was highly homologous to proteose-peptone component 3 (PP3). Since molecular weight of PP3 is 28 kDa, it is suggested that the 19.0 kDa protein is derived from degradation of PP3 during fermentation of fat-free milk. Moreover, PP3 purified from fat-free milk also enhanced IgM production of HB4C5 cells.

Keywords: Abbreviations; BSA; bovine serum albumin; ELISA; enzyme-linked immunosorbent assay; IFN-γ; interferon γ; Ig; immunoglobulin, IL-4, interleukin 4; IPSF; immunoglobulin production-stimulating factor; LPS; lipopolysaccharide; NaPB; sodium phosphate buffer; PBL; peripheral blood lymphocyte(s); PBS; phosphate buffered saline; PP3; proteose-peptone component 3; TNF-α; tumor necrosis factor αFermented milk; Immunoglobulin production; Interferon-gamma; Peripheral blood lymphocytes; Proteose-peptone component 3

Effects of light and chloramphenicol stress on incorporation of nitrogen into cyanophycin in Synechocystis sp. strain PCC 6308 by Mary M. Allen; Courtney Yuen; Lea Medeiros; Nora Zizlsperger; Maliha Farooq; Nancy H. Kolodny (pp. 241-246).

¹H NMR spectroscopy was used to compare the uptake of nitrogen into cyanobacterial cyanophycin from two sources: from the breakdown of intracellular proteins and amino acids, and directly from the external growth medium. Cells grown initially in medium containing¹⁴N-nitrate were transferred to¹⁵N-nitrate medium in the presence of chloramphenicol in both low (4 μE m⁻² s⁻¹) and normal (100 μE m⁻² s⁻¹) light, and in low light alone. Cyanophycin was separated from cells and analyzed by¹H NMR spectroscopy. Cyanophycin is synthesized both from¹⁴N (degradation of cellular proteins) and from¹⁵N in the medium, the latter at a faster rate and to a greater extent under all conditions. SDS-PAGE showed that cyanophycin synthesis takes place by addition of monomers to already synthesized polymer.

Keywords: Cyanobacteria; Cyanophycin; Nitrogen; NMR; Chloramphenicol; Light

In vitro binding of purified NahR regulatory protein with promoter Psal by Hoo Hwi Park; Woon Ki Lim; Hae Ja Shin (pp. 247-255).

The NahR regulatory protein activates the naphthalene catabolic operon through binding to the Psal promoter in the presence of salicylate. Here, we investigated in vitro binding interaction between NahR and Psal using purified functional recombinant NahR. The T7-tagged NahR was shown to exist as a monomer in solution. Electrophoretic mobility shift assay (EMSA) showed that purified NahR bound to Psal in 3 different forms, whereas surface plasmon resonance (SPR) showed on an SPR chip at ratios ranging from 1:1 (at 0.42 μM NahR) to 8:1 (at 6.8 μM NahR). The binding was slightly inhibited by salicylate, suggesting that salicylate may not be involved in the binding of NahR to the promoter, but rather may be important in the activation of prebound NahR. An examination of the binding kinetics by SPR for the interaction between NahR and Psal revealed that the equilibrium dissociation constant was approximately 2.44×10⁻⁶ M and the association and dissociation rates were 7.82×10⁴ M⁻¹ s⁻¹ and 0.191 s⁻¹, respectively. These results demonstrate for the first time that purified NahR binds as a monomer to Psal and undergoes multimerization. In addition, we present novel data on the kinetics of NahR binding.

Keywords: Surface plasmon resonance; Electrophoretic mobility shift assay; Kinetic; NahR regulatory protein; Psal; promoter; Salicylate

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BBA - General Subjects (v.1725, #2)