BBA - General Subjects (v.1725, #1)

The adsorption of condensed tannins (procyanidins) on solid cell wall material was quantified using the Langmuir isotherms formulation. Six tannins fractions differing by their size (number average degree of polymerisation between 2.5 and 65) and composition (presence of galloyl groups from to 0 to 22%, proportions of (+)-catechin to (−)-epicatechin from traces to one CAT for three EPI) were used. Two cell walls differing only by their physical characteristics were obtained by mild or harsh drying, with surface areas of 2.15 and 0.52 m2/g, respectively. The total amounts of procyanidins retained on the cell wall materials increased with their concentrations while the proportions of retained decreased, and a plateau was reached at high concentrations. The apparent affinity of procyanidins for CWM isolated from apples increased with their molecular weight. Decrease of the CWM porosity by harsh drying slightly decreased the apparent affinity and increased the apparent saturation levels when constants were expressed relative to cell wall weight, but strongly increased both apparent affinity and apparent saturation levels per surface units.
Keywords: Tannin; Degree of polymerisation; Galloylation; Porosity; Grape; Pear;

The adsorption of condensed tannins (procyanidins) of varying degrees of polymerisation and percentage of galloylation on solid polysaccharides substrates was quantified using the Langmuir isotherms formulation. Pectins and xyloglucans, which are soluble polysaccharides, were first cross-linked by, respectively, dibromopropane and epichlorohydrin to obtain insoluble covalent gels. Cellulose and starch, being insoluble in the buffer solution at room temperature, were used as bought. Apparent affinity constants obtained for the pure polysaccharides were as follows : pectin ≫ xyloglucan > starch > cellulose. The apparent affinity constants increased with the molecular weight of the procyanidins, except with cellulose. Higher affinities were obtained with pectin, a polysaccharide having the ability to develop a gel-like network, forming hydrophobic pockets able to encapsulate procyanidins. Filamentous and globular polysaccharides, like cellulose and xyloglucan, bound procyanidins weakly. Higher apparent saturation levels were obtained for cellulose and xyloglucans, the arrangement of which would favour cooperativity and stacking. Pectin had lower saturation levels probably due to a steric hindrance effect.
Keywords: Tannin; Degree of polymerisation; Galloylation; Pectin; Xyloglucan; Cellulose; Starch; Cross-linking;

Genetic algorithm for large-scale maximum parsimony phylogenetic analysis of proteins by Tobias Hill; Andor Lundgren; Robert Fredriksson; Helgi B. Schiöth (19-29).
Inferring phylogeny is a difficult computational problem. For example, for only 13 taxa, there are more then 13 billion possible unrooted phylogenetic trees. Heuristics are necessary to minimize the time spent evaluating non-optimal trees. We describe here an approach for heuristic searching, using a genetic algorithm, that can reduce the time required for weighted maximum parsimony phylogenetic inference, especially for data sets involving a large number of taxa. It is the first implementation of a weighted maximum parsimony criterion using amino acid sequences. To validate the weighted criterion, we used an artificial data set and compared it to a number of other phylogenetic methods. Genetic algorithms mimic the natural selection's ability to solve complex problems. We have identified several parameters affecting the genetic algorithm. Methods were developed to validate these parameters, ensuring optimal performance. This approach allows the construction of phylogenetic trees with over 200 taxa in practical time on a regular PC.
Keywords: Genetic algorithm; Phylogeny; G-protein-coupled receptor; Evolution;

A chemiluminescence (CL) method was developed for the evaluation of oxidative damage to biomolecules induced by singlet oxygen (1O2) and for the evaluation of the protective effects of antioxidants. The 1O2 was generated from the reaction of H2O2  + OCl. Results showed that the CL signal from the reaction of H2O2  + OCl was weak, however, it was enhanced dose-dependently with the addition of DNA and unsaturated fatty acid, respectively. Spectra analysis indicated that the enhanced CL could be ascribed to the decay of triplet-excited carbonyl compounds, which were generated from the reaction of 1O2 plus the biomolecules. On the other hand, the enhanced CL produced in the above systems could be effectively inhibited by lycopene, β-carotene, VC, and VE, but could not be inhibited by mannitol, SOD, and NaN3. The mechanism therein was discussed.
Keywords: Antioxidant; Singlet oxygen; Oxidative damage; Chemiluminescence;

The gene cluster containing the nitrile hydratase (NHase) and amidase genes of a moderate thermophile, B. pallidus RAPc8 has been cloned and sequenced. The (5.9 kb) section of cloned DNA contained eight complete open reading frames, encoding (in order), amidase (belonging to the nitrilase related aliphatic amidase family), nitrile hydratase β and α subunits (of the cobalt containing class), a 122-amino acid accessory protein, designated P14K, a homologue of the 2Fe-2S class of ferredoxins and three putative proteins with distinct homology to the cobalt uptake proteins cbiM, cbiN and cbiQ of the S. typhimurium LT2 cobalamin biosynthesis pathway. The amidase and nitrile hydratase genes were subcloned and inducibly expressed in Escherichia coli, to levels of approximately 37 U/mg and 49 U/mg, respectively, without the co-expression of additional flanking genes. However, co-expression of P14K with the NHase structural genes significantly enhanced the specific activity of the recombinant NHase. This is the first description of an accessory protein involved in thermostable NHase expression. Modelling of the P14K protein structure has suggested that this protein functions as a subunit-specific chaperone, aiding in the folding of the NHase α subunit prior to α-β subunit association and the formation of α2β2 NHase holoenzyme.
Keywords: Nitrile hydratase; Bacillus; Thermostable; P14k; Chaperone;

By means of Mono P column chromatography, an effective phosphate acceptor (EPA) of casein kinase 2 (CK2) was purified from the Bowman-Birk-type proteinase inhibitor (BBI) fraction of soybean seeds. The most acidic EPA (aEPA, pI  = approx. 3.7) was heavily phosphorylated when incubated with CK2 and 5 μM [γ-32P]ATP in the presence of poly-Arg (a CK2 activator) in vitro. However, aEPA was slightly phosphorylated by casein kinase 1 (CK1) as effective as C-kinase and not at all by A-kinase in vitro. The 13 N-terminal amino acid residues (SDHSSSDDESSKP) of aEPA were 100% homologous to the corresponding sequence of soybean BBI-type proteinase inhibitor CII (SBI CII). Polyamine at 3 mM stimulated 4.6-fold the CK2-mediated phosphorylation of aEPA, and this phosphorylation was sensitive to quercetin (ID50  = approx. 0.1 μM) in vitro. Furthermore, two basic proteins [Mr = 29,000 (p29) and 17,000 (p17)] copurified with BBI were identified as proteolytic cleavage products of basic 7S globulin and functioned as potent CK2 activators in vitro. aEPA fully phosphorylated by CK2 in the presence of poly-Arg or basic proteins formed a complex with trypsin, whereas unphosphorylated aEPA was digested by trypsin in vitro. These results suggest that (i) aEPA (a BBI isoform) may coexist with two basic proteins (p29 and p17) generated from basic 7S globulin; and (ii) the physiological interaction between aEPA and its binding trypsin-like proteinases may be regulated through specific phosphorylation of aEPA by CK2 activated with the two basic proteins in legume seeds.
Keywords: Casein kinase 2; Effective CK2 substrate; Bowman-Birk-type proteinase inhibitor; Potent CK2 activator; Soybean trypsin inhibitor;

Membranous osteogenesis system modeled with KUSA-A1 mature osteoblasts by Satoshi Matsumoto; Isao Shibuya; Satoshi Kusakari; Kaoru Segawa; Taro Uyama; Akinori Shimada; Akihiro Umezawa (57-63).
Several stromal cells were established from murine bone marrow cultures. One of the KUSA subclones, KUSA-A1 cells, displays osteogenic characteristics in vitro and in vivo. The calcium deposition, osteocalcin release, and parathyroid hormone (PTH) responsiveness of KUSA-A1 cells indicate that they are mature osteoblasts or osteocytes. Bone had formed in subcutaneous tissue 1 week after subcutaneous injection of cells into immunodeficient mice. The osteogenesis by KUSA-A1 was not mediated by chondrogenesis and thus was considered to be membranous ossification. These unique characteristics of KUSA-A1 cells provide an opportunity to analyze the process of membranous ossification in detail.
Keywords: Membranous osteogenesis; Stromal cell; KUSA; Osteoblast; Gap junction;

Both eyes of flatfishes are located on one side of the body due to asymmetrical eye migration. The molecular mechanisms underlying such asymmetry is poorly understood. As an initial step, we have adopted suppression subtractive hybridization for the identification of upregulated genes during metamorphosis involving eye migration in Japanese flounder, Paralichthys olicaceus. One of the upregulated genes was identified as the splicing factor arginine/serine rich-3 (SFRS3). Sequence analysis of SFRS3 revealed that it encodes a protein of 168 amino acids containing the typical eukaryotic RNA recognition motif (RRM) and an arginine/serine-rich region. The overall amino acid sequences of the Japanese flounder SFRS3 was highly conserved with that of other organisms. The expression of flounder SFRS3 gene increased sharply from the beginning of metamorphosis and reached a high level of expression at stage H of metamorphosis 43 days after hatching. The SFRS3 gene upregulation was mainly limited to the head region, particularly in the rapidly proliferative tissues, the lateral ethmoid and “skin thickness” on blind side, which are thought as two proliferative tissues to push the eye movement. In spite of the upregulated expression of SFRS3 during metamorphosis, its role in metamorphosis involving eye migration requires further studies.
Keywords: SFRS3; Fish; Flounder; Metamorphosis; Eye; Development;

A new microperoxidase from Marinobacter hydrocarbonoclasticus by Lorenzo Caputi; Alessandra Di Tullio; Luana Di Leandro; Francesco De Angelis; Francesco Malatesta (71-80).
The preparation and characterization of a new microperoxidase obtained from proteinase K-treated cytochrome c 552 from Marinobacter hydrocarbonoclasticus (previously known as Pseudomonas nautica) are presented. This microperoxidase (MMP-5) has novel structural properties relative to previously reported microperoxidases, as the two intervening amino acid (X) residues within the consensual CXXCH c-type heme binding motif are missing, yielding a heme-pentapeptide with increased solubility in aqueous solvents and a 1–2 order of magnitude higher stability of the monomeric state relative to canonical microperoxidases. The electronic spectra in the near-UV and visible regions have been studied as a function of MMP-5 concentration and pH. The spectroscopic properties of MMP-5 are typical of microperoxidases with high-spin hexa- or pentacoordinate heme species dominant in the 1–8 pH range and low-spin states prevailing at higher pH values. In the presence of hydrogen peroxide, MMP-5 displays peroxidatic activities towards several compounds.
Keywords: Marinobacter hydrocarbonoclasticus; Microperoxidase; Cytochrome c 552; Heme-peptides;

We tested a working hypothesis that stress genes and anti-oxidant enzyme machinery are induced by the organophosphate compound dichlorvos in a non-target organism. Third instar larvae of Drosophila melanogaster transgenic for hsp70 were exposed to 0.1 to 100.0 ppb dichlorvos and 5.0 mM CuSO4 (an inducer of oxidative stress and stress genes) and hsp70, and activities of acetylcholinesterase (AchE), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) product were measured. The study was further extended to examine tissue damage, if any, under such conditions. A concentration- and time-dependent increase in hsp70 and anti-oxidant enzymes was observed in the exposed organism as compared to control. A comparison of stress gene expression with SOD, CAT activities and LPO product under similar experimental conditions revealed that induction of hsp70 precedes the anti-oxidant enzyme activities in the exposed organism. Further, concomitant with a significant inhibition of AChE activity, significant induction of hsp70 was observed following chemical exposure. Mild tissue damage was observed in the larvae exposed to 10.0 ppb dichlorvos for 48 h when hsp70 expression reaches plateau. Dichlorvos at 0.1 ppb dietary concentration did not evoke significant hsp70 expression, anti-oxidant enzymes and LPO and AchE inhibition in the exposed organism, and thereby, was found to be non-hazardous to D. melanogaster. Conversely, 1.0 ppb of the test chemical stimulated a significant induction of hsp70 and anti-oxidant enzymes and significant inhibition of AchE; hence this concentration of test chemical was hazardous to the organism. The present study suggests that (a) both stress genes and anti-oxidant enzymes are stimulated as indices of cellular defense against xenobiotic hazard in D. melanogaster with hsp70 being proposed as first-tier bio-indicator of cellular hazard, (b) 0.1 ppb of the test chemical may be regarded as No Observed Adverse Effect Level (NOAEL), and 1.0 ppb dichlorvos as Low Observed Adverse Effect Level (LOAEL).
Keywords: Dichlorvos; hsp70; SOD; CAT; LPO; AchE; Transgenic Drosophila;

Pig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: Insight in the catalytic mechanisms of this enzyme by Piotr Szyniarowski; Bernard Lakaye; Jan Czerniecki; Alexander F. Makarchikov; Pierre Wins; Ilca Margineanu; Bernard Coumans; Thierry Grisar; Lucien Bettendorff (93-102).
Thiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian 25-kDa thiamine triphosphatase. The enzyme was active in all mammalian species studied except pig, although the corresponding mRNA was present. In order to determine whether the very low ThTPase activity in pig tissues is due to the absence of the protein or to a lack of catalytic efficiency, we expressed human and pig ThTPase in E. coli as GST fusion proteins. The purified recombinant pig GST-ThTPase was found to be 2–3 orders of magnitude less active than human GST-ThTPase. Using site-directed mutagenesis, we show that, in particular, the change of Glu85 to lysine is responsible for decreased solubility and catalytic activity of the pig enzyme. Immunohistochemical studies revealed a distribution of the protein in pig brain very similar to the one reported in rodent brain. Thus, our results suggest that a 25-kDa protein homologous to hThTPase but practically devoid of enzyme activity is expressed in pig tissues. This raises the possibility that this protein may play a physiological role other than ThTP hydrolysis.
Keywords: Thiamine triphosphate; Thiamine triphosphatase; Site-directed mutagenesis; Pig; Brain; Immunohistochemistry;

Antioxidant properties of two gallotannins isolated from the leaves of Pistacia weinmannifolia by Xingyu Zhao; Handong Sun; Aijun Hou; Qinshi Zhao; Taotao Wei; Wenjuan Xin (103-110).
Pistacia weinmannifolia J. Poisson ex Franch (Anacardiaceae) is a shrub or arbor widely found in Yunnan province of China and its leaves are used as traditional Chinese medicine by herbalists. The leaves of P. weinmannifolia are rich in phenolic compounds, among which two novel gallotannins, Pistafolin A and Pistafolin B, are identified. In the present investigation, the antioxidant efficiency of Pistafolin A and Pistafolin B in preventing lipid, protein and DNA from reactive oxygen species-mediated damage was studied. Both Pistafolin A and Pistafolin B inhibited the peroxyl-radical induced lipid peroxidation of l-α-phosphatidylcholine liposomes dose-dependently and prevented the bovine serum albumin from peroxyl-induced oxidative damage. Pistafolin A and Pistafolin B also inhibited copper (II)-1,10-phenanthroline complex-induced DNA oxidative damage. Both Pistafolin A and Pistafolin B scavenged the hydrophilic 2,2′-azinobis(3-ethylbenzothiozoline-6-sulphonic acid) diammonium salt-free radicals and the hydrophobic 1,1-dipheny-2-picrylhydrazyl radicals effectively, suggesting they may act as hydrogen donating antioxidants. The protective effects of the two gallotannins against oxidative damage of biomacromolecules were due to their strong free radical scavenging ability. Pistafolin A with three galloyl moieties showed stronger antioxidant ability than Pistafolin B with two galloyl moieties.
Keywords: Pistacia weinmannifolia; Pistafolin A; Pistafolin B; Gallotannin; Antioxidant;

Oxygen radicals photo-induced by ferric nitrilotriacetate complex by Koichiro Tsuchiya; Kaori Akai; Akira Tokumura; Shinji Abe; Toshiaki Tamaki; Yoshiharu Takiguchi; Kenji Fukuzawa (111-119).
This study examined the photo-induced generation of reactive oxygen species (ROS) by the carcinogenic iron(III)–NTA complex. Iron(III)–NTA complex (1:1) has three conformations (type (a) in acidic conditions of pH 1−6, type (n) in neutral conditions of pH 3−9, and type (b) in basic conditions of pH 7−10) with two pK a values (pK a1  ≈ 4, pK a2  ≈ 8). The iron(III)–NTA complex was reduced to iron(II) under cool-white fluorescent light without the presence of any reducing agent, and the reduction rates of the three conformations of iron(III)–NTA were in the order type (a) > type (n) > type (b) as reported previously (Akai K. et al., Free Radic. Res. 38, 951–962, 2004). ROS generation was investigated by electron paramagnetic resonance (EPR) spectroscopy with a spin-trapping technique. Apparent EPR signals attributed to PBN/· 13CH3 and PBN/·OCH3 spin adducts were observed after incubation of the iron(III)–NTA complex was mixed with α-phenyl-tert-butylnitrone (PBN) and 13C-DMSO in an aerobic condition. The addition of catalase effectively attenuated the PBN adducts, but superoxide dismutase enhanced them. Taken together, these results indicate that the iron(III)–NTA complex is spontaneously reduced to the iron(II)–NTA complex by light under acidic to neutral pH, and in turn transfers an electron to molecular oxygen to form ROS.
Keywords: Nitrilotriacetate; Free radical; Electron paramagnetic resonance; Photochemical; Reduction; Oxidative stress;

Free radicals and other reactive oxygen species (ROS) are generated by all aerobic cells and are widely believed to play a significant role in aging as well as a number of degenerative or pathological diseases. This study compared the free radical-scavenging properties and antioxidant activity of YCP, a polysaccharide from the mycelium of a marine filamentous fungus Phoma herbarum YS 4108 and its two chemically sulfated derivatives YCP-S1 and YCP-S2. Sulfation, which masks hydroxyl groups of YCP polysaccharide molecule, could introduce new antioxidant activity, such as superoxide and hydroxyl radicals scavenging activity, metal chelating action, lipid peroxidation and linoleic acid oxidation inhibition capability. Furthermore, sulfated YCP was more potent than YCP at protecting erythrocytes against oxidative damage hemolysis. The current data suggest for the first time that sulfation of polysaccharide significantly increases its antioxidant activity and the chemical modification of polysaccharides may allow the preparation of derivatives with new properties and a variety of applications.
Keywords: YCP; Polysaccharide; Sulfation; Free radical; Antioxidant;

α1-Acid glycoprotein (AGP), an acute phase reactant, is extensively glycosylated at five Asn-linked glycosylation sites. In a number of pathophysiological states, including inflammation, rheumatoid arthritis, and cancer, alterations of Asn-linked glycans (N-glycans) have been reported. We investigated alteration of N-glycans at each of glycosylation sites of AGP in the sera of patients with acute and chronic inflammation.AGP purified from sera was digested with Glu-C and the liberated glycopeptides were isolated by reverse phase HPLC. N-glycans released with peptide N-glycosidase F and followed by neuraminidase treatment were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.Site-specific differences in branching structures were observed among N-glycosylation sites 1, 3, 4 and 5. Within the sera of patients with acute inflammation, increases in bi-antennary and decreases in tri- and tetra-antennary structures were observed, as well as increases in α1,3-fucosylation, at most glycosylation sites. In the sera of patients with chronic inflammation, increased rates of tri-antennary α1,3-fucosylation at sites 3 and 4 and tetra-antennary α1,3-fucosylation at sites 3, 4 and 5 were detected. Although there were no significant differences between acute and chronic sera in site directed branching structures, significant differences of α1,3-fucosylation were detected in tri-antennary at sites 2, 4 and 5 and in tetra-antennary at sites 3 and 4.Little variation in the N-glycan composition of the glycosylation sites of AGP was observed among healthy individuals, while the sera of patients with acute inflammation demonstrated increased numbers of bi-antennary and α1,3-fucosylated N-glycan structures at each glycosylation site.
Keywords: α1-Acid glycoprotein; Sialyl Lewis X; Inflammation; MALDI-TOFMS; N-glycan;

Cloning, expression and characterization of a 46.5-kDa metallopeptidase from Bacillus halodurans H4 sharing properties with the pitrilysin family by Soumaila Dabonné; Claire Moallic; Jean-Pierre Sine; Sébastien Niamké; Michel Dion; Bernard Colas (136-143).
A 1242 base pair DNA fragment from Bacillus halodurans H4 isolated from alkaline sediments of Lake Bogoria (Kenya) coding for a potential protease was cloned and sequenced. The hexa-histidine-tagged enzyme was overexpressed in Escherichia coli and was purified in one step by immobilized-metal affinity chromatography (IMAC) on Ni-NTA resin. The protease (ppBH4) presents an inverted zincin motif, HXXEH, which defines the inverzincin family. It shares several biochemical and molecular properties with the clan ME family M16 metallopeptidases (pitrilysins), as well as with database hypothetical proteins that are potential M16 family enzymes. Thus, like insulysin and nardilysin, but contrary to bacterial pitrilysin, ppBH4 is inactivated by sulfhydryl alkylating agents. On the other hand, like bacterial pitrilysin, ppBH4 is sensitive to reducing agents. The enzymatic activity of ppBH4 is limited to substrates smaller than proteins. In contrast to insulin, dynorphin and insulin B-chain are very good substrates for ppBH4 and several cleavage sites are common with those observed with well-characterized pitrilysins. As deduced from amino acid sequence, as well as determined by gel-filtration and SDS-polyacrylamide gel electrophoresis, ppBH4 is an active monomer of 46.5 kDa. This feature distinguishes ppBH4 from all other enzymes of the pitrilysin family so far described whose molecular masses range from 100 to 140 kDa.
Keywords: Pitrilysin; Metallopeptidase; Inverzincin; Dynorphin; Insulin B-chain; Bacillus halodurans;