BBA - General Subjects (v.1672, #3)

The ability of numerous diverse compounds and ions to cross the bacterial cytoplasmic membrane by diffusion and active transport is highly dependent on cytoplasmic membrane fluidity, which can be measured using fluorescent probes to estimate membrane polarization values. However, membrane polarization data are lacking for most bacterial species. The cytoplasmic membrane polarization values for Arthrobacter sp. ATCC 21908, Bacillus cereus NRC 3045, Pseudomonas fluorescens R2F, Pseudomonas putida NRC 2986 and Escherichia coli C600 bacterial cells were spectrofluorometrically measured over a temperature range from 10 to 50°C, and in the absence and presence of 1 μg/ml tetracycline, using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to obtain new information on their membrane fluidity. At an assay temperature of 10°C, E. coli cells grown in the absence of tetracycline exhibited the highest cytoplasmic membrane polarization value (least fluid membrane) of 0.446, followed by values of 0.392, 0.371, 0.344 and 0.293, respectively, for B. cereus, Arthrobacter sp., P. fluorescens and P. putida. At an assay temperature of 30°C, the polarization values ranged from 0.357 to 0.288 for cells grown in the absence of tetracycline, regardless of the species. B. cereus grown in the presence of 1 μg/ml tetracycline had lower polarization values than when grown in the absence of this antibiotic at all assay temperatures. Regardless of the absence or presence of 1 μg/ml tetracycline in the growth medium, all bacterial species generally exhibited a more fluid membrane as the assay temperature increased from 10 to 50°C. To our knowledge, these are some of the first cytoplasmic membrane polarization values reported for these Gram-negative and Gram-positive bacteria over a broad temperature range and also for cells grown in the presence of tetracycline.
Keywords: Bacteria; Cytoplasmic membrane; 1,6-Diphenyl-1,3,5-hexatriene (DPH); Fluorescent probe; Gram-negative; Gram-positive; Membrane fluidity; Membrane polarization; Tetracycline;

High level of vitamin B12 was incorporated into kaiware daikon (Japanese radish sprout) by soaking its seeds in B12 solutions. Vitamin B12 amount incorporated into kaiware daikon increased up to 1.5 μg/g wet sprout with the soaking time of seeds in 0–200 μg/ml B12 solution. Vitamin B12 could be extracted more from the sample heated for a short time than from that of control without heat treatmetnt.
Keywords: Kaiware daikon (Japanese radish sprout); Vitamin B12; Vitamin B12 in vegetable; Cobalamin in vegetable;

The aim of this study was to develop the cell microarray that allows efficient transfer of multiple genes into mammalian cells cultured on the microarray in a high-throughput fashion. A microarray was fabricated using a gold-coated glass plate having a micropatterned, self-assembled monolayer of alkanethiols carrying ionic and nonionic terminal groups. Plasmid DNA and a cationic lipid were loaded by alternate electrostatic adsorption to the microspots to obtain a plasmid DNA microarray. The loading and the release of lipid-DNA complex were studied by, respectively, the fluorescence staining of DNA and the imaging of the microarray with a surface plasmon resonance (SPR) apparatus. The transfection efficiency was evaluated by directly plating and culturing human embryonic kidney cells onto the microarray. The results demonstrated that cells which adhered to the DNA-loaded spots were transfected to express the encoded model proteins for several days. The chemistry of the monolayers and the number of alternate adsorption cycles had large effects on the efficiency of transfection. This may be explained from the availability of the lipid–DNA complex to the cells directly contacted. We conclude that the micropatterned, self-assembled monolayers greatly facilitate regionally defined loading of DNAs and expression of the encoded protein in mammalian cells.
Keywords: Cell microarray; Gene transfection; High-throughput analysis; Micropatterning; Self-assembled monolayer; DNA loading;

Characterization of the cytotoxic mechanism of Mana-Hox, an analog of manzamine alkaloids by Lan Chun Tu; Chen-Kung Chou; Ching-Yeu Chen; Yao-To Chang; Ya-Ching Shen; Sheau-Farn Yeh (148-156).
Mana-Hox is a synthetic analog of manzamines, which are β-carboline alkaloids isolated from marine sponges. Mana-Hox exhibited cytotoxicity against various tumor cell lines with the IC50 range from 1 to 5 μM. Cell cycle synchronization and flow cytometric analysis showed that Mana-Hox delayed cell cycle progression at mitosis. At the concentration that delayed mitotic progression, bipolar spindle with lagged chromosomes and multipolar spindle with disorganized chromosomes were detected. The presence of such aberrant mitotic cells accompanied by the activation of spindle checkpoint that delayed cells exit from mitosis. However, after a short delay, lagged chromosomes were able to display in the abnormal metaphase plates, and subsequent cell division resulting in chromosome missegregation. Furthermore, the aberrant mitotic cells showed lower viability, indicating that Mana-Hox-induced cell death resulting from chromosome missegregation. This study is the first to explore cytotoxic mechanism of a manzamine-related compound and understand its potential as a lead compound for the development of future anticancer agents.
Keywords: Mana-Hox; Manzamine; Cytotoxicity; Aberrant mitosis; Chromosome missegregation; Anticancer agent;

Expression of cell surface Lewis X and Y antigens and FUT4 mRNA is increased in Jurkat cells undergoing apoptosis by Yutaro Azuma; Miyuki Ito; Akiyoshi Taniguchi; Kojiro Matsumoto (157-163).
Cell surface molecules undergo specific changes during apoptosis, including the expression of phosphatidylserine (PS) and some proteins and alterations in sugar chains. Among the various sugar chains on the cell surface, Lewis X (LeX) and Lewis Y (LeY) antigens are key determinants for a variety of biological processes.We studied the changes in LeX and LeY expression in Jurkat cells, a human T cell line, during apoptosis. Flow cytometry showed that LeX and LeY antigen expression was enhanced on the cell surface during apoptosis induced by anti-Fas antibody. To clarify the mechanism of enhanced LeX and LeY expression, we assessed the expression levels of fucosyltransferase (FUT1, 2, 3-5-6, 4, and 9) mRNAs that are predominantly expressed in Jurkat cells and which are considered to form LeX and LeY. The expression of FUT4 mRNA was up-regulated after exposing cells to anti-Fas antibody. Moreover, the increase in LeX and LeY antigen levels was significantly suppressed by caspase 3 or 8 inhibitors. These results indicated that the induction of FUT (mainly FUT4), the gene expression of which is mediated by signals downstream of caspase 3, increases LeX and LeY expression in apoptotic cells.
Keywords: Apoptosis; Fas; Caspase; Lewis antigen; Fucosyltransferase;

Oxygen binding and oxidation reactions of human hemoglobin conjugated to carboxylate dextran by Yiping Jia; Francine Wood; Patrick Menu; Béatrice Faivre; Alexis Caron; Abdu I Alayash (164-173).
Human hemoglobin (Hb) conjugated to benzene tetracarboxylate substituted dextran produces a polymeric Hb (Dex-BTC-Hb) with similar oxygen affinity to that of red blood cells (P 50=28–29 mm Hg). Under physiological conditions, the oxygen affinity (P 50) of Dex-BTC-Hb is 26 mm Hg, while that of native purified human HbA0 is 14 mm Hg, but it exhibits a slight reduction in cooperativity (n 50), Bohr effect, and lacks sensitivity to inositol hexaphosphate (IHP), when compared to HbA0. Oxygen-binding kinetics, measured by rapid mixing stopped-flow method showed comparable oxygen dissociation and association rates for both HbA0 and Dex-BTC-Hb. The rate constant for NO-mediated oxidation of the oxy form of Dex-BTC-Hb, which is governed by NO entry to the heme pocket, was reduced to half of the value obtained for HbA0. Moreover, Dex-BTC-Hb is only slightly more sensitive to oxidative reactions than HbA0, as shown by about 2-fold increase in autoxidation, and slightly higher H2O2 reaction and heme degradation rates. Dextran-BTC-based modification of Hb produced an oxygen-carrying compound with increased oxygen release rates, decreased oxygen affinity and reduced nitric oxide scavenging, desirable properties for a viable blood substitute. However, the reduction in the allosteric function of this protein and the lack of apparent quaternary T→R transition may hinder its physiological role as an oxygen transporter.
Keywords: Hemoglobin; Oxygen affinity; Redox reaction; Blood substitute;

A single amino acid substitution can shift the optimum pH of DNase I for enzyme activity: biochemical and molecular analysis of the piscine DNase I family by T Yasuda; H Takeshita; R Iida; M Ueki; T Nakajima; Y Kaneko; K Mogi; Y Kominato; K Kishi (174-183).
We purified four piscine deoxyribonucleases I (DNases I) from Anguilla japonica, Pagrus major, Cryprus carpio and Oreochromis mossambica. The purified enzymes had an optimum pH for activity of approximately 8.0, significantly higher than those of mammalian enzymes. cDNAs encoding the first three of these piscine DNases I were cloned, and the sequence of the Takifugu rubripes enzyme was obtained from a database search. Nucleotide sequence analyses revealed relatively greater structural variations among the piscine DNase I family than among the other vertebrate DNase I families. From comparison of their catalytic properties, the vertebrate DNases I could be classified into two groups: a low-pH group, such as the mammalian enzymes, with a pH optimum of 6.5–7.0, and a high-pH group, such as the reptile, amphibian and piscine enzymes, with a pH optimum of approximately 8.0. The His residue at position 44 of the former group is replaced by Asp in the latter. Replacement of Asp44 of piscine and amphibian DNases I by His decreased their optimum pH to a value similar to that of the low-pH group. Therefore, Asp44His might be involved in an evolutionarily critical change in the optimum pH for the activity of vertebrate DNases I.
Keywords: cDNA cloning; Deoxyribonuclease I; Molecular evolution; Optimum pH; Purification; Fish;

Activation and inhibition of Candida rugosa and Bacillus-related lipases by saturated fatty acids, evaluated by a new colorimetric microassay by Cristian Ruiz; Serena Falcocchio; Entela Xoxi; F.I Javier Pastor; Pilar Diaz; Luciano Saso (184-191).
Research on lipase inhibitors could help in the therapy of diseases caused by lipase-producing microorganisms and in the design of novel lipase substrate specificities for biotechnology. Here we report a fast and sensitive colorimetric microassay that is low-cost and suitable for high-throughput experiments for the evaluation of lipase activity and inhibition. Comparison of Candida rugosa activity and inhibition with previous HPLC results validated the method, and revealed the importance of the reaction mixture composition. The assay was used to evaluate the effect of saturated fatty acids on Bacillus-related lipases. Cell-bound esterases were strongly inhibited by fatty acids, suggesting a negative feedback regulation by product, and a role of these enzymes in cell membrane turnover. Bacillus subtilis LipA was moderately activated by low concentrations of fatty acids and was inhibited at greater concentrations. LipB-like esterases were highly activated by myristic and lauric acids and were only slightly inhibited by high capric acid concentrations. Such an activation, reported here for the first time in bacterial lipases, seems to be part of a regulatory system evolved to ensure a high use of carbon sources, and could be related to the successful adaptation of Bacillus strains to nutrient-rich environments with strong microbial competition.
Keywords: Lipase; Colorimetric; Saturated fatty acid; Bacillus;

Non-covalent interaction between procyanidins and apple cell wall material by C Le Bourvellec; S Guyot; C.M.G.C Renard (192-202).
The adsorption of procyanidins on cell wall material were quantified by bringing into contact a solution of procyanidins and a suspension of cell wall material. The influence of structural features such as degree of polymerisation (DP) and percentage of galloylation (% gall), and of physico-chemical parameters such as pH, ionic strength, temperature and presence of ethanol were investigated.The amount of procyanidins bound to the cell wall increased with the DP, the % gall, and the proportion of (+)-catechin, the last indicating an effect of the stereochemistry of the flavan-3-ols.Complex formation between procyanidins and cell wall material was not affected by pH in the range 2.2–7 but it was decreased by urea, dioxane and ethanol. Adsorption increased with increasing ionic strength and decreased with increasing temperature. This indicated that the bonds which governed the interaction between procyanidins and cell wall material were weak energy bonds of the type hydrogen bond and hydrophobic interaction.
Keywords: Tannin; Polysaccharide; Adsorption; Apple; Cider; Grape;

Iron release, superoxide production and binding of autologous IgG to band 3 dimers in newborn and adult erythrocytes exposed to hypoxia and hypoxia-reoxygenation by Lucia Ciccoli; Viviana Rossi; Silvia Leoncini; Cinzia Signorini; Julian Blanco-Garcia; Carlo Aldinucci; Giuseppe Buonocore; Mario Comporti (203-213).
Iron is released in a desferrioxamine (DFO)-chelatable form when erythrocytes are challenged by an oxidative stress. The release is increased when an accelerated removal of erythrocytes occurs such as in perinatal period, in which iron release is greater in hypoxic than in non-hypoxic newborns. This suggests that an hypoxic environment at birth promotes iron release. To test this possibility, iron release in a model of hypoxia, hypoxia-reoxygenation and normoxia was studied in newborn and adult erythrocytes. In newborn erythrocytes, hypoxia induced a much greater iron release compared to an equal period of normoxia. In adult erythrocytes, hypoxia also induced a greater iron release as compared to normoxia, but it was much lower than that seen with newborn erythrocytes. Methemoglobin (MetHb) formation roughly paralleled iron release. The phenylhydrazine-promoted superoxide anion (O2) production was greater with normoxic but lower with hypoxic erythrocytes from newborns as compared to that from adults. This discrepancy between iron release and O2 production may be explained by the shift towards MetHb in hemoglobin autoxidation. Iron diffusion out of the erythrocytes was much higher with hypoxic erythrocytes from newborns as compared to that from adults. Also the binding of autologous IgG to band 3 dimers (AIgGB) is much greater with hypoxic erythrocytes from newborns as compared to that from adults, suggesting that the level of iron release is related to the extent of band 3 clustering and that hypoxia accelerates removal of erythrocytes from bloodstream in in vivo condition.
Keywords: Iron release; Oxidative stress; Hypoxic erythrocyte; Free radical; Band 3 dimer; Newborn;

Overexpression, purification and characterization of the acidic ribosomal P-proteins from Candida albicans by Dariusz Abramczyk; Marek Tchórzewski; Dawid Krokowski; Aleksandra Boguszewska; Nikodem Grankowski (214-223).
In all eukaryotic cells, acidic ribosomal P-proteins form a lateral protuberance on the 60S ribosomal subunit—the so-called stalk—structure that plays an important role during protein synthesis. In this work, we report for the first time a full-length cloning of four genes encoding the P-proteins from Candida albicans, their expression in Escherichia coli, purification and characterization of the recombinant proteins. Considerable amino acid sequence similarity was found between the cloned proteins and other known fungal ribosomal P-proteins. On the basis of their phylogenetic relationship and amino acid similarity to their yeast counterparts, the C. albicans P-proteins were named P1A, P1B, P2A and P2B. Using three different approaches, namely: chemical cross-linking method, gel filtration and two-hybrid system, we analyzed mutual interactions among the C. albicans P-proteins. The obtained data showed all the four P-proteins able to form homo-oligomeric complexes. However, the ones found between P1B–P2A and P1A–P2B were dominant forms among the C. albicans P-proteins. Moreover, the strength of interactions between particular proteins was different in these two complexes; the strongest interactions were observed between P1B and P2A proteins, and a significantly weaker one between P1A and P2B proteins.
Keywords: Acidic ribosomal protein; Candida albicans; Expression; P-protein interaction;