BBA - General Subjects (v.1670, #1)

Photodynamic inhibition of enzymatic detachment of human cancer cells from a substratum by Anatoly Uzdensky; Asta Juzeniene; Li-Wei Ma; Johan Moan (1-11).
Photodynamic therapy (PDT) is currently used for cancer treatment. It is shown that sublethal PDT of human WiDr adenocarcinoma cells and D54Mg glioblastoma cells with 5-aminolevulinic acid (ALA), disulfonated tetraphenylporphyrine (TPPS2a), or MitoTracker Red (MTR) inhibits their trypsin-induced detachment from a plastic substratum. TPPS2a was bound selectively to the plasma membrane, whereas MTR was found in mitochondria. Both granular and diffuse fluorescence of ALA-derived protoporphyrin IX (PpIX) was observed in the perinuclear cytoplasm but not in the plasma membrane of WiDr cells stained for 2 h with 1 mM ALA. In D54Mg cells, PpIX fluorescence was observed not only in the cytoplasm but also in the plasma membrane. Fluorescence measurements showed a progressive accumulation of PpIX in the WiDr cells during incubation with ALA and a PpIX efflux into the medium after 1 h or longer incubation. PpIX retained in the plasma membrane during efflux may be responsible for PDT-induced impairment of cell adhesion. On the other hand, MTR-PDT or ALA-PDT after 15-min incubation, when the newly synthesized PpIX should remain in mitochondria, also inhibited enzymatic cell detachment. Therefore, photodynamic targeting of mitochondria, remote from the cell surface where adhesion occurs, may disturb cell adhesion. Photodynamic inhibition of enzymatic cell detachment may be related to PDT-induced inhibition of tumour metastasis.
Keywords: ALA-PDT; MitoTracker Red; TPPS2a; Cell adhesion; Photosensitizer localization; Detachment;

The purpose of this study was to determine if high ascorbate of the human aqueous protects the lens against oxidative stress. Previous studies with the rat lens have been inconclusive because of its fortification with aldose reductase (AR), an important antioxidant. The human lens is deficient in this activity. These studies were hence done with the mouse lens, a species deficient in this enzyme. The reactive oxygen species (ROS)-induced physiological damage to the tissue was assessed in organ culture, by measuring its ability to actively transport 86Rb+ ions, in the absence and presence of ascorbate. In addition, the status of tissue metabolism and its antioxidant reserve were assessed by quantitating ATP and glutathione (GSH). As expected, ROS decreased the membrane transport activity as well as the levels of ATP and GSH. Ascorbate minimized these toxic effects substantially. The presence of high ascorbate, therefore, appears highly beneficial in protecting the lens against oxidative damage and cataract formation, despite a deficiency of AR. The findings therefore appear to be significant from the point of view of using this nutrient for delaying the onset of cataract development in human beings, therapeutically as well as nutritionally.
Keywords: Oxidative stress; Cataract; Ascorbate; Na+-K+-ATPase; Rubidium transport;

Enzymatic and nonenzymatic formation of reactive oxygen species from 6-anilino-5,8-quinolinequinone by Toyoshi Hasegawa; Atsushi Bando; Koichiro Tsuchiya; Shinji Abe; Masumi Okamoto; Kazuyoshi Kirima; Satoru Ueno; Masanori Yoshizumi; Hitoshi Houchi; Toshiaki Tamaki (19-27).
The nonenzymatic and enzymatic formation of reactive oxygen species (ROS) from LY83583 (6-anilino-5,8-quinolinequinone) was investigated by electron paramagnetic resonance (EPR) spectroscopy. In the presence of thiol compounds such as glutathione and l-cysteine, LY83583 underwent a one-electron reduction due to low redox potential (−0.3±0.01 V vs. SCE), followed by formation of LY83583 semiquinone anion radical. This species was characterized by EPR spectroscopy under an argon atmosphere at neutral pH. Under an aerobic condition, this species interacts with molecular oxygen to form a superoxide anion radical. GSH-conjugated LY83583 was also identified by NMR and FAB-MS. When LY83583 was applied to PC12 cells, ROS formation was completely inhibited by both the flavoenzyme inhibitor DPI and the DT-diaphorase inhibitor dicumarol. On the other hand, ROS generation occurred independent of intracellular GSH level. These results indicate that LY83583 can generate ROS both enzymatically and nonenzymatically, although the enzymatic formation is dominant over the nonenzymatic system in PC12 cells.
Keywords: Free radical; LY83583; Electron paramagnetic resonance; PC12 cell; Glutathione; Oxidative stress;

Reactivity of flavonoids with 1-hydroxyethyl radical: a γ-radiolysis study by Abdelghafour Marfak; Patrick Trouillas; Daovy P. Allais; Claude A. Calliste; Jeanne Cook-Moreau; Jean-Luc Duroux (28-39).
We have investigated the reactivity between 11 flavonoids and 1-hydroxyethyl radical (HER). HER was recently implicated in many liver injuries induced by ethanol intoxication. In this study, HER was generated by radiolysis; due to its reaction rate, HER is well known to be responsible for solute degradation in irradiated ethanol. Flavonoid ethanol solutions were irradiated with γ-rays and the flavonoid degradation was followed by HLPC. We observed the degradation of flavonols while all other flavonoids (flavones, flavanones, dihydroflavonols, catechins) were not degraded after irradiation. The major radiolysis products were identified by NMR and LC-MS and we concluded that flavonols were essentially transformed into depsides. We proposed a reactivity mechanism between flavonols and HER. In a first step, H-transfer occurred from the 3-OH group to HER. Afterwards, C-ring opening occurred due to the presence of the 2,3-double bond in flavonols. Finally, we calculated the reaction constants in order to evaluate the antioxidant activity of flavonols against HER and to compare it with reference compounds.
Keywords: Flavonoid; 1-Hydroxyethyl radical (HER); γ-Radiolysis; Free radical scavenging;

Modulation of porphyrin binding to serum albumin by pH by Pavel Kubát; Kamil Lang; Pavel Anzenbacher (40-48).
In this study, we show that the difference in acidity of functional groups in porphyrin photosensitizers provides a meaningful avenue to achieve differential localization and retention of porphyrins in tissues and cells, and in the end could be a positive factor in the photodynamic treatment of cancer (PDT). We have demonstrated that meso-tetraphenylporphyrin derivative with four phosphonate (P(O)(OH)2) moieties exists in aqueous solutions mainly in four forms that differ by a degree of protonation of the porphyrin ring and ionization of the phosphonate group. It is shown that each porphyrin form has different affinities toward the model protein (bovine serum albumin, BSA). Thus pH of the medium significantly modulates the affinity of the phosphonate porphyrin toward BSA. At lower pH (pH 6.0), the phosphonate porphyrin and BSA form a complex with affinity constant of K b=6.9×105 M−1, while at pH 7.0 the K b=6.1×105 M−1. At pH 8.0 the association is significantly lower. Because cancerous cells have generally lower pH (pH∼6.9) compared to healthy cells (pH∼7.4), the pH of such cells could be a decisive factor for cellular retention of the porphyrin in the form of an associate with intracellular proteins. Moreover, we have also demonstrated that the protonation/deprotonation equilibria do not negatively affect the photophysical properties or ability of phosphonate porphyrin to generate singlet oxygen.
Keywords: Tetraphenylporphyrin; Protein; Association; Phosphonate; Singlet oxygen; Photodynamic therapy;

Amino acid transport is down-regulated in ischemic human intestinal epithelial cells by Masafumi Wasa; Hong-Sheng Wang; Yoshiyuki Shimizu; Akira Okada (49-55).
Amino acid transport across the plasma membrane is essential for supplying enterocytes with amino acids for cellular metabolism. We studied amino acid transport during ischemic conditions using human intestinal epithelial cell line Caco-2. Cells were incubated under nutrient-deprived (phosphate-buffered saline, PBS), hypoxic, and ischemic (PBS+hypoxia) conditions. Ischemia resulted in a significant decrease in glutamine transport by a mechanism that decreased V max without affecting K m. The expression of system ATB° (glutamine transporter) mRNA decreased in the ischemic and nutrient-deprived groups, suggesting that the down-regulation of glutamine transport is due to modification of expression of the ATB° gene. The transport of glutamate and leucine, DNA synthesis, and intracellular glutathione also decreased in the ischemic group. These findings throw some light on the mechanism of intestinal epithelial damage during ischemia.
Keywords: Intestinal ischemia; Caco-2 cell; Glutamine; Amino acid transport; System ATB°;

Nuclear localization of tetrahydrobiopterin biosynthetic enzymes by Lina Elzaouk; Stephanie Laufs; Dirk Heerklotz; Walter Leimbacher; Nenad Blau; Annette Résibois; Beat Thöny (56-68).
Biosynthesis of the tetrahydrobiopterin (BH4) cofactor, essential for catecholamines and serotonin production and nitric oxide synthase (NOS) activity, requires the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). Upon studying the distribution of GTPCH and PTPS with polyclonal immune sera in cross sections of rat brain, prominent nuclear staining in many neurons was observed besides strong staining in peri-ventricular structures. Furthermore, localization studies in transgenic mice expressing a Pts-LacZ gene fusion containing the N-terminal 35 amino acids of PTPS revealed β-galactosidase in the nucleus of neurons. In contrast, PTPS-β-galactosidase was exclusively cytoplasmic in the convoluted kidney tubules but nuclear in other parts of the nephron, indicating again that nuclear targeting may occur only in specific cell categories. Furthermore, the N terminus of PTPS acts as a domain able to target the PTPS-β-galactosidase fusion protein to the nucleus. In transiently transfected COS-1 cells, which do not express GTPCH and PTPS endogenously, we found cytoplasmic and nuclear staining for GTPCH and PTPS. To further investigate nuclear localization of all three BH4-biosynthetic enzymes, we expressed Flag-fusion proteins in transiently transfected COS-1 cells and analyzed the distribution by immunolocalization and sub-cellular fractionation using anti-Flag antibodies and enzymatic assays. Whereas 5–10% of total GTPCH and PTPS and ∼1% of total SR were present in the nucleus, only GTPCH was confirmed to be an active enzyme in nuclear fractions. The in vitro studies together with the tissue staining corroborate specific nuclear localization of BH4-biosynthetic proteins with yet unknown biological function.
Keywords: Tetrahydrobiopterin; Nuclear localization; Monoamine neurotransmitter; Aromatic amino acid monooxygenase; NOS;

Role of the W07-toxin on Vibrio cholerae-induced diarrhoea by Shalmoli Bhattacharyya; Sujata Ghosh; Jasleen Shant; Nirmal K. Ganguly; Siddhartha Majumdar (69-80).
Vibrio cholerae W07 strain isolated from a cholera epidemic in South India, lacked the ctx gene but could still secrete a novel toxin, the W07-toxin that could cause fluid accumulation in ligated rabbit ileal loop. The important intracellular messengers implicated in this study were Ca2+, cyclic AMP, inositol triphosphate and protein kinase C (PKC). A number of inhibitors/channel blockers have further shown the major role of [Ca2+]i in modulation of the toxin-induced cellular response. An increase in the level of reactive oxygen species (ROS) in the W07-toxin-stimulated enterocytes correlated with the decrease in the levels of antioxidant enzymes, catalase and superoxide dismutase (SOD). The reactive nitrogen intermediates (RNI) detected by measuring the levels of nitrite and citrulline, were found to be high in the enterocytes triggered with the W07-toxin, thereby indicating their role in toxin-mediated change in mucosal permeability. The precise role of the toxin has also been authenticated by conducting the experiments with W07-toxin preincubated in the presence of IgGWT (IgG isolated from antitoxin sera) or GM1. Thus, a significant increase in the levels of second messengers and a decrease in antioxidant defenses appear to be important in mediating the fluid secretion caused by this novel toxin from V. cholerae W07.
Keywords: Vibrio cholerae; W07-toxin; Intracellular messenger;

Genetic instability in EBV-transformed lymphoblastoid cell lines by Aisha Mohyuddin; Qasim Ayub; Saima Siddiqi; Denise R. Carvalho-Silva; Kehkashan Mazhar; Sadia Rehman; Sadaf Firasat; Ahsana Dar; Chris Tyler-Smith; S. Qasim Mehdi (81-83).
Epstein Barr virus (EBV)-transformed lymphoblastoid cell lines are commonly used to provide an inexhaustible supply of DNA. We examined microsatellite instability in these cell lines in 35 individuals where DNA was available from the original blood samples and from cultured cell lines. Mutations were observed in 0.3% of the analyses, thus providing a quantitative measure of somatic mutation rate.
Keywords: EBV transformation; Lymphoblastoid cell line; Microsatellite; Population genetic; STR;

Molecular cloning and expression analysis of novel wheat cysteine protease by Akihiko Tsuji; Manami Tsuji; Hiromiti Takami; Shingo Nakamura; Yoshiko Matsuda (84-89).
A cDNA clone encoding a novel papain-like cysteine protease was isolated from wheat germ (Triticum aestivum). This cDNA encoded a 371-residue protein, designated WCP2, composed of signal peptide followed by a propeptide and a mature protease containing active site residues that are highly conserved among the papain family. The mature WCP2 protein (26 kDa) was detected in the quiescent embryo and its level of expression in the germinating embryo was greatly increased.
Keywords: Cysteine protease; Embryo; Germ; Germination; Papain; Wheat;