BBA - General Subjects (v.1623, #2-3)

Role of sialic acid and sulfate groups in cervical mucus physiological functions: study of Macaca radiata glycoproteins by Nasir-ud-Din; Daniel C. Hoessli; Elisabeth Rungger-Brändle; Syed Altaf Hussain; Evelyne Walker-Nasir (53-61).
The influence of charged groups in glycoproteins was investigated to assess their effect on the physiological functions of bonnet monkey cervical mucus. The macromolecular glycoproteins from peri-ovulatory, midcycle phase cervical mucus were treated with Pronase, trypsin and chymotrypsin and the enzyme-resistant glycoproteins purified by gel filtration on Sepharose 4B and a high molecular weight component containing carbohydrates, proteins and sulfate groups was recovered in high yield. This material still reacted with an antiserum directed against purified midcycle glycoprotein but not against another antiserum directed against luteal phase purified glycoproteins. Upon treatment with Pronase, trypsin and chymotrypsin, asialoglycoproteins and desulfated asialoglycoproteins released fragments of low molecular sizes, none of which reacted with the anti-midcycle glycoprotein antiserum. Cervical mucus collected from the estrogenic phase displayed a morphology supporting sperm migration, and this mucus retains the same morphology and reacts with the anti-midcycle glycoprotein antiserum following mild treatment with sialidase and subsequently with Pronase. These results imply that charged carbohydrate groups help maintain the structural and functional integrity of the mucus glycoprotein in its biological environment.
Keywords: Cervical mucus; Sialic acid; Sulfate group; Mucin and glycoprotein; Bonnet monkey;

Transacetylations to carbohydrates catalyzed by acetylxylan esterase in the presence of organic solvent by Peter Biely; Ken K.Y. Wong; Ian D. Suckling; Silvia Špániková (62-71).
Various conditions were applied to test the ability of acetylxylan esterase (AcXE) from Schizophyllum commune to catalyze acetyl group transfer to methyl β-d-xylopyranoside (Me-β-Xylp) and other carbohydrates. The best performance of the enzyme was observed in an n-hexane-vinyl acetate-sodium dioctylsulfosuccinate (DOSS)–water microemulsion at a molar water–detergent ratio (w 0) of about 4–5. Although the enzyme was found to have a half-life of about 1 h in the system, more than 60% conversion of Me-β-Xylp to acetylated derivatives was achieved. Under identical reaction conditions, the enzyme acetylated other carbohydrates such as methyl β-d-cellobioside (Me-β-Cel), cellotetraose, methyl β-d-glucopyranoside (Me-β-Glcp), 2-deoxy-d-glucose, d-mannose, β-1,4-mannobiose, -mannopentaose, -mannohexaose, β-1,4-xylobiose and -xylopentaose. This work is the first example of reverse reactions by an acetylxylan esterase and a carbohydrate esterase belonging to family 1.
Keywords: Acetylxylan esterase; Acetylation; Mode of action; Chemoenzymic synthesis; Low-water-content media;

Characterization of the major allergens purified from the venom of the paper wasp Polistes gallicus by Barbara Pantera; Donald R. Hoffman; Lara Carresi; Gianni Cappugi; Stefano Turillazzi; Giampaolo Manao; Maurizio Severino; Igino Spadolini; Giuseppe Orsomando; Gloriano Moneti; Luigia Pazzagli (72-81).
Allergic reactions to vespid stings are one of the major causes of IgE-mediated anaphylaxis. Vespa and Vespula venoms are closely related; Polistes venom is more distantly related and its allergens are less well studied. There is limited cross-reactivity between Polistes and the other vespid venoms because of differences in the epitopes on the allergen molecules.In this study, the major allergens of Polistes gallicus are isolated and characterized. P. gallicus venom contains four major allergens: phospholipase, antigen 5 (Ag5), hyaluronidase and protease that were characterized by mass spectrometry and specific binding to IgE. The complete amino acid sequence of Ag5 and the sequence of the N-terminal region of phospholipase were also determined. The alignment of Ag5 from P. gallicus (European species) and Polistes annularis (American species) shows an 85% identity that increases to 98% within the same subgenus. This could suggest the presence of specific epitopes on Ag5 molecule being the variations on the superficial loops. The features of the P. gallicus allergens could explain the partial cross-reactivity found between the American and European Polistes venoms, and suggest that the use of European Polistes venoms would improve the diagnostic specificity and the therapy of European patients and of North American patients sensitized by European Polistes.
Keywords: Allergen; Venom; Polistes; Purification; Amino acid sequence; Antigen 5;

1,5-Anhydroglucitol stimulates insulin release in insulinoma cell lines by Toshikazu Yamanouchi; Tae Inoue; Kaoru Ichiyanagi; Tadashi Sakai; Nobuyuki Ogata (82-87).
Concentrations of 1,5-anhydroglucitol (1,5-AG), which is a major circulating polyol, decrease in patients with diabetes mellitus. In both insulinoma-derived RINr and MIN6 cells, 1,5-AG stimulated insulin release within the range of 0.03–0.61 mM in a dose-dependent manner. Insulin release was maximally stimulated by 1,5-AG to levels that reached 25% and 100% greater than that of control (1,5-AG-free group) in RINr and MIN6 cells, respectively. A physiological concentration of 1,5-AG stimulated insulin release after a 5-min incubation and this action was maintained for 60 min. In addition, at approximately 1/200 the concentration of glucose, 1,5-AG had additive action with 20 mM glucose. The action of 1,5-AG on insulin secretion with other types of saccharides and polyol was similarly additive. Mannnoheptulose and diazoxide suppressed the stimulative action of 1,5-AG on insulin release. The secretagogue action of 1,5-AG seemed to be independent on an increase in the intracellular content of cAMP and ATP. These results suggest that 1,5-AG can stimulate insulin secretion through a mechanism that completely differs from that of glucose.
Keywords: 1,5-Anhydroglucitol; Saccharide; Secretagogue; Insulin secretion; MIN6 cell;

The effect of glycation on the structure, function and biological fate of human serum albumin as revealed by recombinant mutants by Keisuke Nakajou; Hiroshi Watanabe; Ulrich Kragh-Hansen; Toru Maruyama; Masaki Otagiri (88-97).
Recombinant wild-type human serum albumin (rHSA), the single-residue mutants K199A, K439A and K525A and the triple-residue mutant K199A/K439A/K525A were produced using a yeast expression system. Portions of the rHSA were glycated to different degrees (2.5–250 mM d-glucose). As detected by far-UV and near-UV CD, intrinsic tryptophan-fluorescence and probed by 1,1′-bis(4-anilino)naphthalene-5,5-disulfonic acid, the single-residue mutations had no effect on albumin conformation, whereas the triple-residue mutation and glycation caused conformational changes. The triple-residue mutation and glycation had comparable increased effects on high-affinity binding of warfarin (site I), but decreased effects on high-affinity binding of dansylsarcosine (site II) and the esterase-like activity of albumin. The relation between plasma half-lives in rats were found to be glycated rHSA (50 mM glucose)<triple-residue mutated rHSA<rHSA. The opposite trend was found for liver and kidney uptakes in mice. Even though the functional and the in vivo properties of rHSA could be effected differently by the minor conformational changes caused by the triple-residue mutation and glycation, the present findings indicate that the effect of glycation can be partly explained by blockage of the positive charges of lysine at positions 199, 439 and 525.
Keywords: Human serum albumin; Glycation; Positive charge; Organ uptake; Site-directed mutagenesis;

Nitric oxide (NO) is a short lived, readily diffusible intracellular messenger molecule associated with multiple organ-specific regulatory functions. In this communication, we elucidate the effect of exogenous NO administration, using nitroglycerin (GTN), on ferric nitrilotriacetate (Fe-NTA)-induced renal oxidative stress, hyperproliferative response and necrosis in ddY mice. Fe-NTA is a known complete renal carcinogen as well as renal and hepatic tumor promoter, which act by generating oxidative stress in the tissues. GTN treatment to ddY mice prior to Fe-NTA administration resulted in a highly significant protection against Fe-NTA-induced renal oxidative stress, hyperproliferative response and necrosis. In oxidative stress protection studies, the decrease in the level of renal glutathione and antioxidant enzyme activities induced by Fe-NTA were significantly reversed by GTN pretreatment in a dose-dependent manner (12–46% recovery, P<0.05–0.001). GTN pretreatment also resulted in a dose-dependent inhibition (24–39% inhibition, P<0.05–0.001) of Fe-NTA-induced lipid peroxidation as measured by TBARS formation in renal tissues. Similarly, in hyperproliferation protection studies, GTN pretreatment showed a strong inhibition of Fe-NTA-induced renal ornithine decarboxylase (ODC) activity (51–57% inhibition, P<0.001) and [3H]thymidine incorporation (43–58% inhibition, P<0.001) into renal DNA. GTN pretreatment almost completely prevented kidney biomolecules from oxidative damage and protected the tissue against the observed histopathological alterations. From this data, it can be concluded that exogenously produced NO from GTN might scavenge reactive oxygen species (ROS) and decreases toxic metabolites of Fe-NTA and thereby inhibiting renal oxidative stress. In addition, exogenously produced NO can also inhibit Fe-NTA-induced hyperproliferative response by down-regulating the activity of ODC and the rate of [3H]thymidine incorporation into renal DNA and could be suggested as another possible clinical application for this NO-donor (GTN, traditionally used as a vasodilator) in oncological medicine.
Keywords: Nitroglycerin; Antioxidant enzyme; Tumor promotion; Oxidative stress; Ferric nitrilotriacetate; Renal tubular necrosis; Lipid peroxidation;

The formation of 4-hydroxy-5-methyl-3(2H)-furanone (HMF, norfuraneol) by spinach ribosephosphate isomerase was reinvestigated [Arch. Biochem. Biophys. 202(1) (1980) 106]. Incubation experiments using d-ribose-5-phosphate and d-ribulose-5-phosphate clearly revealed a spontaneous nonenzymatic formation of the hydroxy-furanone from the ketose-phosphate under physiological conditions at 35 °C and pH 7.5, whereupon up to 1.3% of d-ribulose-5-phosphate was transformed to HMF within 15 h. 4,5-Dihydroxy-2,3-pentanedione was deduced as ultimate precursor of HMF, since addition of o-phenylenediamine to the incubation mixture led to lower amounts of HMF and to the formation of 3-(1,2-dihydroxyethyl)-2-methylquinoxaline, which was identified by means of high pressure liquid chromatography with diode array detection (HPLC–DAD), HPLC–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS/MS) and NMR spectroscopy. Additionally, the spontaneous formation of 4,5-dihydroxy-2,3-pentanedione was demontrated by its conversion to the respective alditol acetate using either NaBH4 or NaBD4 for the reduction. Comparative gas chromatography–mass spectrometry (GC–MS) analysis revealed the incorporation of two deuterium atoms and confirmed the dicarbonyl structure. Application of 1-13C-d-ribulose-5-phosphate as well as 5-13C-d-ribulose-5-phosphate and analysis of the derived quinoxaline derivatives by HPLC–ESI–MS/MS demonstrated the formation of the methyl-group at C-5 of the carbohydrate phosphate in consequence of a nonenzymatic phosphate elimination. Application of o-phenylenediamine into ripe tomatoes led to the detection of 3-(1,2-dihydroxyethyl)-2-methylquinoxaline by means of HPLC–MS/MS analysis implying the genuine occurrence of 4,5-dihydroxy-2,3-pentanedione in this fruit.
Keywords: d-Ribulose-5-phosphate; 4,5-Dihydroxy-2,3-pentanedione; 4-Hydroxy-5-methyl-3(2H)-furanone; Quinoxaline; Tomato;

Suppression of apolipoprotein AI gene expression in HepG2 cells by TNF α and IL-1β by Michael J Haas; Mohammad Horani; Amjad Mreyoud; Brian Plummer; Norman C.W Wong; Arshag D Mooradian (120-128).
Plasma inflammatory cytokines are elevated in obese subjects as well as in those with type 2 diabetes. This presumably results in systemic insulin resistance, characterized by a pro-atherogenic plasma lipid profile and reduced apolipoprotein AI (apoAI) protein levels. To determine how cytokine-mediated insulin resistance suppresses apoAI gene expression, we investigated the effect of tumor necrosis factor α (TNF α) and interleukin-1β (IL-1β) on apoAI protein, mRNA, and transcriptional activity in the human hepatoma cell line HepG2. ApoAI secretion was suppressed in a dose-dependent manner in HepG2 cells treated with both cytokines. ApoAI protein levels were 2892±22.0, 2263±117, 2458±25.0, 3401±152, 2333±248, 1520±41.5 and 956.0±11.0 arbitrary units (AU) in cells treated with 0, 0.3, 1.0, 3.0, 10, 30, and 100 ng/ml TNF α, achieving statistical significance in the 30 and 100 ng/ml range (P<0.0009). ApoAI protein levels were 4055±360, 3697±101, 3347±327, 1561±33.0, 1581±182, 810.0±59.5, and 1766±717 AU in cells treated with similar doses of IL-1β, achieving statistical significance within the range of 3–100 ng/ml (P<0.02). ApoAI mRNA levels were suppressed 50.8% in HepG2 cells treated with 30 ng/ml TNF α for 24 h (P<0.05), and remained suppressed for up to 96 h. Similarly, treatment of cells with 30 ng/ml IL-1β for 24 h, resulted in 42.9% reduction in apoAI mRNA levels (P<0.05) and remained suppressed for up to 96 h.In order to determine if the effect of TNF α and IL-1β occurs at the transcriptional level, HepG2 cells were transfected with a chloramphenicol acetyltransferase (CAT) reporter gene plasmid containing the full-length apoAI promoter, and after 24 h, treated with TNF α (30 ng/ml), IL-1β (30 ng/ml), or both cytokines. CAT activity was suppressed by both cytokines (24.0±1.9% acetylation in control cells vs. 5.6±1.2% (P<0.0004), 10.2±1.5% (P<0.0006), and 3.9±0.9% acetylation (P<0.0002) in cells treated with TNF α, IL-1β, and the combination of both cytokines, respectively) suggesting that cytokine-mediated suppression occurs at the transcriptional level. Using a series of apoAI deletion constructs, the cytokine response element was mapped between nucleotides −325 and −186 (relative to the transcriptional start site). This region contains a previously identified and characterized cis-element, site A, which binds several different transcription factors. Finally, electrophoretic mobility shift assays (EMSA) showed that TNF α treatment of HepG2 cells is associated with reduced nuclear factor binding to site A.These studies suggest that inflammatory cytokines down-regulate apoAI expression at least partly through inhibition of binding of the nuclear factors to site A of the apoAI promoter.
Keywords: TNF α; IL-1β; Insulin resistance; Apolipoprotein AI;

Chemoattractant-stimulated calcium influx in Dictyostelium discoideum does not depend on cGMP by Douwe M. Veltman; Jan Sietse De Boer; Peter J.M. Van Haastert (129-134).
Chemoattractant stimulation of Dictyostelium cells leads to the opening of calcium channels in the plasma membrane, causing extracellular calcium to flux into the cell. The genetically uncharacterised mutants stmF and KI8 show strongly altered chemoattractant-stimulated cGMP responses. The aberrant calcium influx in these strains has provided evidence that the chemoattractant-stimulated calcium influx is potentiated by cGMP. We have tested this hypothesis in genetically defined mutants by measuring the calcium influx in a strain that lacks intracellular cGMP due to the disruption of two guanylyl cyclases, and in a strain with increased cGMP levels caused by the disruption of two cGMP-degrading phosphodiesterases. The results reveal that the calcium influx stimulated by cAMP or folic acid is essentially identical in these strains. We conclude that cGMP is not involved in chemoattractant-stimulated calcium influx.
Keywords: Calcium influx; Cyclic GMP; Streamer F; Dictyostelium;

Expression changes in mRNAs and mitochondrial damage in lens epithelial cells with selenite by P.B. Belusko; T. Nakajima; M. Azuma; T.R. Shearer (135-142).
An overdose of sodium selenite induces cataracts in young rats. The mid-stage events producing the cataract include calpain-induced hydrolysis and precipitation of lens proteins. Apoptosis in lens epithelial cells has been suggested as an initial event in selenite cataracts. Expression levels of two genes associated with apoptosis were altered in lens epithelial cells from selenite-injected rats. The purpose of the present experiment was to perform a more comprehensive search for changes in expression of mRNAs in lens epithelial cells in order to more fully delineate the early events in selenite-induced cataracts. Lens epithelial cells were harvested at 1 and 2 days after a single subcutaneous injection of sodium selenite (30 μmol/kg body weight) into 12-day-old rats. Gene expression was analyzed using a commercial DNA array (Rat Genome U34A GeneChip array, Affymetrix). Of approximately 8000 genes assayed by hybridization, 13 genes were decreased and 27 genes were increased in the rat lens epithelial cells after injection of selenite. Some of the up-regulated genes included apoptosis-related genes, and a majority of the down-regulated genes were mitochondrial genes. Previously observed changes in expression of EGR-1 mRNA were also confirmed. Changes in the expression patterns of mRNAs were also confirmed by RT-PCR. To determine the mechanism for damage of lens epithelial cells (alpha TN4 cell) by culture in selenite, leakage of cytochrome c from mitochondria was measured. Selenite caused significant leakage of cytochrome c into the cytosol of alpha TN4 cells. Our data suggested that the loss of integrity of lens epithelial cells by selenite might be caused by preferential down-regulation of mitochondrial RNAs, release of cytochrome c, and impaired mitochondrial function. Up-regulation of mRNAs involved in maintenance of DNA, regulation of metabolism, and induction of apoptosis may also play roles.
Keywords: Selenite; Mitochondria; DNA microarray; Lens epithelium;

The N-terminal domain of NrtC, the ATP-binding subunit of nitrate/nitrite ABC-transporter in the cyanobacterium Phormidium laminosum, has been expressed in Escherichia coli as a histidine-tagged fusion protein (His6NrtC1). Binding of ATP to the pure His6NrtC1 was characterized using the nucleotide analogue TNP-ATP [2′(3′)-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate]. Fluorescence assays showed that His6NrtC1 specifically binds Mg2+ TNP-ATP with high affinity, binding being dependent on protein concentration. The presence of ATP prevents the covalent modification of His6NrtC1 by fluorescein 5′-isothiocyanate (FITC), suggesting that this probe reacts at the nucleotide-binding site of NrtC. The active form of the truncated NrtC is a dimer that shows high affinity for TNP-ATP (K d=0.76±0.1 μM). Evidence for the presence of two nucleotide-binding sites per dimer protein is given. Our results indicate that nucleotide binding is strongly dependent on the dimerization of NrtC and that the N-terminal domain of the protein contains the binding site for ATP. No ATPase activity catalyzed in vitro by the truncated subunit was detected.
Keywords: ABC transporter; Nitrate transport; Nucleotide binding; NrtC; Phormidium laminosum; TNP-ATP;