BBA - General Subjects (v.1673, #3)

Influence of colloid, preservation medium and trimetazidine on renal medulla injury by Carole Doucet; Delphine Dutheil; Isabelle Petit; Keqiang Zhang; Michel Eugene; Guy Touchard; Anne Wahl; Francois Seguin; Serge Milinkevitch; Thierry Hauet; Gerard Mauco (105-114).
In organ transplantation, preservation injury is an important factor which could influence short-term and long-term graft outcome. The renal medulla is particularly sensitive to oxidant stress and ischemia–reperfusion injury (IRI). Using an autotransplant pig kidney model, we investigated renal function and medullary damage determined between day 1 and week 2 after 24- or 48-h cold storage in different preservation solutions: University of Wisconsin solution (UW), Hopital Edouard Herriot solution (a high Na+ version of UW), ECPEG (high Na+ preservation solution with PEG) and ICPEG (a high K+ version of ECPEG) with or without trimetazidine (TMZ). TMZ improved renal preservation and increased renal function when added in each preservation solution (particularly HEH and ECPEG). Medullary damage led to the early appearance of trimethylamine-N-oxide (TMAO) followed by 1H-NMR in urine and plasma. TMZ and ECPEG is the most efficient association to reduce medullary damage. This study clarifies the role of colloid and polarity solution and the role of mitochondrial protection by TMZ.
Keywords: Cold preservation; Ischemia–reperfusion injury; Pig kidney model; Trimetazidine; Medulla injury; High-Na+ solution; Kidney inflammation; Organ preservation; Acute tubular necrosis; Organ preservation;

Pioglitazone induces plasma platelet activating factor-acetylhydrolase and inhibits platelet activating factor-mediated cytoskeletal reorganization in macrophage by Chinuyo Sumita; Makiko Maeda; Yasushi Fujio; Jooyeon Kim; Junko Fujitsu; Soji Kasayama; Isamu Yamamoto; Junichi Azuma (115-121).
Platelet activating factor (PAF) is a key molecule for inflammation. To examine a role of peroxisome proliferator-activated receptor gamma (PPARγ) in inflammatory reactions of atherosclerosis, we investigated the effects of 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2) and pioglitazone, PPARγ ligands, on plasma PAF-acetylhydrolase (PAF-AH) expression in THP-1 macrophages. PAF-AH mRNA and protein were up-regulated by the PPARγ ligands. Prostaglandin F2α (PGF2α), a PARγ inhibitor, abrogated the up-regulation of PAF-AH mRNA by pioglitazone, suggesting that PPARγ activation is involved in the induction of PAF-AH by pioglitazone. As PAF promotes the cell motility with cytoskeletal reorganization, we investigated the effect of pioglitazone on PAF-mediated morphological changes in THP-1 macrophages. In the absence of pioglitazone, PAF promoted the elongation of actin cytoskeleton, which was inhibited by pretreatment with pioglitazone. In contrast, pioglitazone was not able to inhibit the morphological changes induced by C-PAF, a non-hydrolyzable PAF agonist. Thus, it is suggested that PAF-induced morphological changes could be inhibited by pioglitazone through PAF-AH, which rapidly hydrolyzed PAF. These data propose that PPARγ/PAF-AH pathway is a clinical target for the prevention against atherosclerosis.
Keywords: PAF-AH; PPARγ; Macrophage; Cytoskeletal reorganization; Atherosclerosis;

Characterization of lectin aggregates in the hemolymph of freshwater prawn Macrobrachium rosenbergii by Ali Pereyra; Roberto Zenteno; Lorena Vázquez; Salvador Martı́nez-Cairo; Adela Rodrı́guez; Guillermo Mendoza-Hernández; Edgar Zenteno; Concepción Agundis (122-130).
In invertebrates, lectins play relevant roles in innate immunity; however, their regulatory mechanisms have not been identified yet. In this work, we purified, by gel filtration and affinity chromatography, lectin aggregates circulating in the hemolymph of the freshwater prawn Macrobrachium rosenbergii and compared their physicochemical properties with a previously described lectin (MrL). High-molecular weight MrL aggregates (MrL-I) lack hemagglutinating activity and showed bands of 62.1, 67.1 and 81.4 kDa, whereas MrL-III, which corresponds to MrL, showed hemagglutinating activity and is constituted by a single 9.6-kDa band as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. MrL-I and MrL-III showed similar amino acid composition but different carbohydrates concentration. Edman degradation indicated NH2-terminal sequence of five amino acids for the 9.6-kDa MrL-III (DVPLL/A) and eleven for the main 81.4-kDa band identified in MrL-I (DVPLL/AXKQQQD); analysis by MALDI-TOF indicated a different tryptic pattern for MrL-I and MrL-III. MrL-I was recognized by monoclonal antibodies against MrL-III. Circular dichroism indicated that the secondary structure in both proteins is similar and contains 23% of β-sheet and 24% of α-helix. Our results suggest that differential posttranslational processes that favor aggregation are involved in regulating the activity of the lectin.
Keywords: Lectin; Hemolymph; Macrobrachium rosenbergii;

Peptides binding to a Gb3 mimic selected from a phage library by Yoshiko Miura; Yuuki Sasao; Masamichi Kamihira; Akio Sakaki; Shinji Iijima; Kazukiyo Kobayashi (131-138).
Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides.
Keywords: Phage display peptide library; Protein–oligosaccharide interaction; Gb3; Shiga toxin; Quartz crystal microbalance;

Preservation of the native protein structure and biological activity in dry protein/excipient mixtures has been previously attributed to either the glass forming properties of the additives or to their ability to hydrogen bond to the protein. There is evidence that both processes are important but it has not yet been elucidated which is the limiting factor that determines the efficiency of a given molecule as a protectant.In this work, gravimetric measurements together with enzyme activity assays have been employed to investigate the protection of proteins by sugars, through direct interaction via hydrogen bonding and as the result of glass formation. As a model protein, trypsin has been employed and the modes of action of two similar disaccharides, sucrose and trehalose, which offer different levels of protection, evaluated and compared. Data obtained on freeze-dried formulations indicate that protein and sugars interact through hydrogen bonding to protein hydration sites. The extent of interaction is found to change dramatically at elevated temperatures; sucrose showing a significantly decreased, and trehalose a considerably increased level of interaction. Protein preservation is shown to be directly related to the number of hydrogen bonds formed. Possible reasons why trehalose interacts more extensively with the protein than sucrose are discussed in terms of differences in the anhydrous structures and molecular mobilities of the sugar molecules.
Keywords: Protein preservation; Trehalose; Sucrose; Protein hydration; Glass transition; Hydrogen bonding;

We have constructed a metabolic model describing the H2O2 elimination by mammalian cells. It comprises three compartments (medium, cytosol, and peroxisome) separated by cytoplasmic and peroxisomal membranes, and H2O2 moves across the membranes with different permeation rate constants. Catalase localizes to peroxisomes, while glutathione peroxidase (GPx) and GSH recycling system (glutathione reductase (GR) and the oxidative pentose phosphate pathway (PPP)) localize to cytosol. The rates of individual enzyme reactions were computed using the experimentally determined activities and rate equations known for mammalian enzymes. Using the model, the concentration dependence of H2O2 elimination rate was obtained by numerical simulation and was compared with experimental data obtained previously with cultured mammalian cells (fibroblasts, human umbilical vein endothelial cells (HUVEC), and PC12 cells). The model was shown to be able to reproduce the data well by assuming appropriate values for the permeability rate constants. The H2O2 permeability coefficients thus estimated for cytoplasmic and peroxisomal membranes were in the same order of magnitude, except that the value for cytoplasmic membrane of PC12 cell was significantly smaller. The results suggest that the membrane permeability is one of the rate-limiting factors in the H2O2 elimination by mammalian cells. Using the model and estimated parameter values, we have examined the rate-limiting enzyme of the metabolic system, as well as the intracellular H2O2 concentration under steady-state and non-steady-state conditions.
Keywords: Hydrogen peroxide; Metabolic model; Catalase; Glutathione peroxidase; PC12 cell; Endothelial cell;

Effects of interleukin (IL) on intracellular free Ca2+ concentration ([Ca2+]i) rise and catecholamine (CA) release were examined in isolated, cultured bovine adrenal chromaffin cells. IL-1α and IL-1β inhibited the rise of [Ca2+]i and CA release induced by acetylcholine (ACh) and excess KCl both in normal and in Ca2+-sucrose medium. Pretreatment by IL-1 receptor antagonist (IL-1RA) blocked the inhibitory actions of IL-1α. IL-1α reduced CA release induced by veratridine in normal medium but not in the presence of diltiazem. Analysis using specific blockers for voltage-operated Ca2+ channels (VOCC) revealed that IL-1α and IL-1β specifically inhibited the P/Q-type Ca2+ channel to reduce [Ca2+]i rise induced by excess KCl. IL-1 did not affect [Ca2+]i rise induced either by bradykinin or caffeine in Ca2+-deprived medium or via activation of store-operated Ca2+ channel (SOC). The inhibitory effects of IL-1α were blocked by pretreatments with herbimycin A, U0126 and PD 98054, but not with SB202190, SP 600125 or pertussis toxin (PTX).These results demonstrated that IL-1 inhibits stimulation-evoked [Ca2+]i rise and CA release in chromaffin cells by blocking voltage-operated P/O-type Ca2+ channels. The inhibitory action of IL-1 may be mediated through the tyrosine kinase and MEK/ERK pathways.
Keywords: IL-1; Voltage-dependent Ca2+ channel; P/Q-type Ca2+ channel; Catecholamine release; Ca2+; Adrenal chromaffin cell;

Interaction of mushroom tyrosinase with aromatic amines, o-diamines and o-aminophenols by Beata G a ̨ sowska; Paweł Kafarski; Hubert Wojtasek (170-177).
3-Amino-l-tyrosine was found to be a substrate of mushroom tyrosinase, contrary to what had previously been reported in the literature. A series of amino derivatives of benzoic acid were tested as substrates and inhibitors of the enzyme. 3-Amino-4-hydroxybenzoic acid, 4-amino-3-hydroxybenzoic acid and 3,4-diaminobenzoic acid were oxidized by this enzyme, as previously reported for Neurospora crassa tyrosinase, but 4-aminobenzoic acid and 3-aminobenzoic acid were not. Interestingly, 3-amino-4-hydroxybenzoic acid was oxidized five times faster than 4-amino-3-hydroxybenzoic acid, confirming the importance of proton transfer from the hydroxyl group at C-4 position. All compounds inhibited the monophenolase activity but their effect on the diphenolase activity was small or negligible. 3-Amino-4-hydroxybenzoic acid was a stronger inhibitor than 4-amino-3-hydroxybenzoic acid, indicating their different binding affinity to the oxy form of the enzyme. Both, however, were weaker inhibitors than 3-amino-l-tyrosine, 4-methoxy-o-phenylenediamine and 3,4-diaminobenzoic acid, which was the strongest inhibitor from among the compounds tested. These results show that the relative positioning of the amino group and the hydroxy group in o-aminophenols with respect to the side chain is important both for binding to the dicopper center and for catalysis.
Keywords: Tyrosinase; o-phenylenediamine; o-aminophenol; Proton transfer;

The effect of xylosides on the synthesis of [35S]-sulfated glycosaminoglycans by endothelial cells in culture was investigated. Ortho-nitrophenyl-β-d-xylose (10-3M) produces a dramatic enhancement on the synthesis of heparan sulfate and chondroitin sulfate secreted to the medium (20- and 100-fold, respectively). Para-nitrophenylxyloside, at the same concentration, produces an enhancement of only 37- and 3-fold of chondroitin sulfate and heparan sulfate, respectively. These differences of action seem to be related with the higher lipophilic character of ortho-nitrophenyl-xyloside. A lower enhancement of the synthesis of the two glycosaminoglycans is also observed with 2-naphtol β-d-xylose and cis/trans-decahydro-2-naphtol β-d-xylose. Besides stimulating the synthesis, O-nitrophenyl-β-d-xylose as PMA [J. Cell. Biochem. 70 (1998) 563] also inhibits [3H]-thymidine incorporation by quiescent endothelial cells stimulated for growth by fetal calf serum (FCS). The combination of xylosides with PMA produced some cumulative effect. PMA stimulates the synthesis of heparan sulfate mainly at G1 phase whereas the highest enhancement of synthesis produced by the xylosides is in the S phase of the endothelial cell cycle.
Keywords: Heparan sulfate and growth factors; Heparan sulfate and nitrophenylxylosides; Cell cycle and xylosides; Inhibition of DNA synthesis and o-nitrophenylxyloside; Xylosides and heparan sulfate synthesis;

Evaluation of active recombinant catalytic domain of human ErbB-2 tyrosine kinase, and suppression of activity by a naturally derived inhibitor, ZH-4B by Xiao-Ning Guo; Li Zhong; Xiu-Hua Zhang; Wei-Min Zhao; Xiong-Wen Zhang; Li-Ping Lin; Jian Ding (186-193).
Human cancers frequently express high levels of ErbB-2 tyrosine kinase, which is associated with aggressive tumor behavior and poor prognosis. ErbB-2 is thus a promising target for cancer therapy. Here we express the catalytic domain of ErbB-2 as a soluble active kinase, and investigate the correlations between its activity and kinase concentration, ATP concentration, substrate concentration and divalent cation type. A simple and effective screening model is established to identify and evaluate potential inhibitors of ErbB-2 kinase. ZH-4B, a naturally derived small molecule compound that potently inhibits ErbB-2 kinase activity with an IC50 value of 2.45±0.56 μM, is identified. In SK-OV-3 human ovarian cancer cells and SK-BR-3 human breast carcinoma cells, ZH-4B blocks epidermal growth factor (EGF)-induced phosphorylation of ErbB-2 in a dose-dependent manner. Our data collectively indicate that ZH-4B is a potential novel anti-cancer agent that deserves further investigation.
Keywords: ErbB-2; Tyrosine kinase inhibitor; Anti-cancer drug; ZH-4B;

Expression of erbB receptors mRNA in thyroid tissues by Seiya Kato; Takako Kobayashi; Kouji Yamada; Kazuhiro Nishii; Hirohide Sawada; Hiroshi Ishiguro; Mitsuyasu Itoh; Hiroomi Funahashi; Akio Nagasaka (194-200).
ErbB family members, such as epidermal growth factor receptor 1 (erbB1), erbB2, erbB3 and erbB4, are widely distributed in organ tissues, and these receptors are suspected tumorigenesis factors.We measured erbB mRNA in thyroid tissues of benign and malignant thyroid tumors or Graves' disease using Genescan. ErbB2 is associated with aggressive cancers and is used as a biological marker for the disease; Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses have shown it to be increased in Graves' disease. Additional studies indicated a similar result in papillary carcinoma cells; mRNAs of erbB2 and erbB3 were increased but erbB4 mRNA was decreased, suggesting distorted erbB expression may be associated with tumorigenesis. However, only erbB2 overexpression is associated with Graves' disease. These data further implicate transmembrane-type receptors in tumorigenesis in the thyroid.
Keywords: Expression; erbB; Thyroid;

Regulation of transcription and activity of Rhizobium etli glutaminase A by Alejandro Huerta-Saquero; Arturo Calderón-Flores; Andrea Dı́az-Villaseñor; Gisela Du Pont; Socorro Durán (201-207).
The present study determines the regulatory mechanisms that operate on Rhizobium etli glutaminase A. glsA gene expression levels were evaluated under several metabolic conditions by fusions of the glsA gene promoter and the transcriptional reporter cassette uidA2-aad. glsA expression was directly correlated to the glutaminase A activity found under the tested growth conditions, reaching its maximum level in the presence of glutamine and during exponential growth phase. Glutamine induces glsA expression.The influence of allosteric metabolites on glutaminase A activity was also determined. The purified enzyme was inhibited by 2-oxoglutarate and pyruvate, whereas oxaloacetate and glyoxylate modulate it positively. Glutaminase A is not inhibited by glutamate and is activated by ammonium.Glutaminase A participates in an ATP-consuming cycle where glutamine is continually degraded and resynthesized by glutamine synthetase (GS). GS and glutaminase A activities appear simultaneously during bacterial growth under different metabolic conditions and their control mechanisms are not reciprocal. Slight overproduction in glutaminase A expression causes a reduction in growth yield and a dramatic decrease in bacterial growth.We propose a model for regulation of glutaminase A, and discuss its contribution to glutamine cycle regulation.
Keywords: Glutamine; Glutaminase A; Glutamine synthetase; Rhizobium etli;

Many insects display non-homeostatic regulation over blood sugar level. The concentration of trehalose varies dramatically depending on physiological and nutritional state. In the absence of dietary carbohydrate, blood trehalose in larvae of the lepidopteran insect Manduca sexta is maintained by gluconeogenesis and is dependent on dietary protein consumption. In the present study, the effect of dietary fat on the glucogenic response of insects to increased dietary protein was examined by NMR analysis of (2-13C)pyruvate metabolism. Last instar larvae were maintained on a carbohydrate-free chemically defined artificial diet having variable levels of casein with and without corn oil. Gluconeogenic flux, the ratio of the rate of gluconeogenesis to the rate of glycolysis, was estimated from the 13C distribution in trehalose arising by gluconeogenesis and the 13C enrichment of alanine due to pyruvate cycling. Insects grew well on carbohydrate-free diets and growth increased with increasing dietary protein level. At all dietary protein levels, larvae grew better on diets with fat. Without dietary fat, larvae were glucogenic but displayed low blood trehalose concentrations, <30 mM, regardless of protein consumption. When fat was included in the diet, however, gluconeogenic flux and blood trehalose level increased sharply in response to increased dietary protein level, with trehalose concentrations >50 mM at higher levels of protein consumption. When offered a choice of a high carbohydrate and a high protein diet, larvae maintained on diets with fat displayed a food preference related to blood sugar level. Those with low blood sugar fed on carbohydrate, while those with high blood sugar preferred protein. Trehalose synthesized from (2-13C)pyruvate exhibited asymmetry in the 13C distribution in individual glucose molecules, indicating a disequilibrium at the triose phosphate isomerase-catalyzed step of the gluconeogenic pathway. In trehalose from larvae on diets with fat, the asymmetric 13C distribution was higher than in trehalose from insects on diets lacking fat. This may partially result from isotopic disequilibrium when unenriched glycerol is metabolized to dihydroxyacetone phosphate following fat hydrolysis. The asymmetry in 13C distribution, however, also occurred in insects on diets without fat and decreased with increased gluconeogenic flux suggesting that true disequilibrium between the triose phosphates is the principal reason for the asymmetry.
Keywords: Gluconeogenesis; Dietary fat; 13C; Invertebrate; NMR; Manduca sexta; Nutrition; Trehalose;