BBA - General Subjects (v.1571, #1)

An investigation has been performed into the biophysical properties of the enveloped mammalian virus, herpes simplex virus type 1 (HSV-1). The dielectrophoretic behaviour of the virus particles was measured as a function of applied frequency (over the range 100 kHz–20 MHz) and conductivity of the suspending medium (over the range 1–100 mS m−1). The dielectric properties of the virus were determined from the dielectrophoretic data using the smeared-out shell model. The data suggest that the intact particle has a surface conductance of 0.3 nS, an internal and membrane permittivity of 75ε o and 7.5ε o, respectively, an internal conductivity of approximately 0.1 S m−1 and a zeta potential of 70 mV.The dielectric properties were measured for intact, fresh virus particles and also for particles following exposure to various modifying agents, such as treatment with enzymes, ionophores and ageing. It is shown that the observed changes in the dielectrophoretic spectrum, and the variations in the dielectric properties of the virus concur with the expected physiological effects of these agents.
Keywords: Herpes simplex virus type 1; Virion; Dielectrophoresis; AC electrokinetics;

Enzyme activities along the tryptophan-nicotinic acid pathway in alloxan diabetic rabbits by Eugenio Ragazzi; Carlo V.L Costa; Laura Caparrotta; Monica Biasiolo; Antonella Bertazzo; Graziella Allegri (9-17).
Recent data from our laboratory have indicated that the rabbit is a suitable animal model for the study of enzyme activities of the tryptophan-nicotinic acid pathway. We report here the pattern of tryptophan metabolism in rabbits made diabetic with alloxan treatment, and hypercholesterolemic with a high-cholesterol diet. A group of rabbits with only hypercholesterolemia was also considered. The enzymes assayed were: liver tryptophan 2,3-dioxygenase (TDO), intestine indoleamine 2,3-dioxygenase (IDO), liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase.TDO showed a reduction of specific activity in liver of diabetic-hyperlipidemic and hyperlipidemic rabbits compared to controls. Intestine IDO activities and liver and kidney kynurenine monooxygenase were unchanged with respect to controls.Kynurenine-oxoglutarate transaminase and kynureninase activities were reduced in the kidneys, but not in the liver, of diabetic-hyperlipidemic rabbits.The main finding was the reduction of 3-hydroxyanthranilate 3,4-dioxygenase activity (expressed as activity per g of fresh tissue) in the liver and kidneys of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared to controls. Conversely, aminocarboxymuconate-semialdehyde decarboxylase activity was significantly higher in diabetic hypercholesterolemic rabbits in comparison with control and hypercholesterolemic rabbits.These data demonstrate that also in diabetic rabbits there is an alteration of tryptophan metabolism at the level of 3-hydroxyanthranilic acid→nicotinic acid step. Also dyslipidemia seems to be involved in enzyme activity variations of the tryptophan metabolism along the kynurenine pathway.
Keywords: Tryptophan metabolism; Kynurenine-pathway enzyme; Alloxan-diabetic rabbit;

A ganglioside of unknown structure (ganglioside X) was purified from chicken brain at embryonic day 12 (E12) and characterized for its structure. Ganglioside X was reactive with a monoclonal antibody A2B5 and migrated below GH1c on thin-layer chromatography (TLC). Extensive treatment of ganglioside X with Clostridium perfringens sialidase produced a single ganglioside product. This ganglioside was identified as GM1 based upon its chromatographic mobility and reactivity to cholera toxin B subunit and anti-GM1 antibody. Partial hydrolysis of ganglioside X by sialidase generated several degradation products including GH1c, GP1c, and GQ1c. Electrospray ionization (ESI)-mass spectrometry (MS) of the permethylated derivative of ganglioside X produced a triple-charged parent ion peak at m/z 1355, which corresponded with the gangliotetraose oligosaccharide structure having seven sialic acids and ceramide with the molecular mass of 566 (as non-methylated form). Collision-induced dissociation (CID)-MS2 showed fragment ions including those at m/z 1066 and 1931; these two ions matched the structures of (NeuAc)3-Gal-Glc-Cer and (NeuAc)4-Gal-GalNAc, respectively. These structures were confirmed by CID-MS3 of the corresponding peaks. Based upon these findings, the structure of ganglioside X was identified as NeuAc-NeuAc-NeuAc-NeuAc-Galβ1–3GalNAcβ1–4(NeuAc-NeuAc-NeuAcα2–3)Galβ1–4Glcβ1-1′Cer. This ganglioside was designated as GS1c. A developmental study demonstrated that GS1c was expressed in chicken brain during a period from E6 to E13 and thereafter decreased rapidly in its concentration. The present study suggests that GS1c may play a specific role in early development of chicken brain.
Keywords: Glycolipid; Ganglioside; C-series ganglioside; A2B5; Chicken; Brain; Development;

NMR structure of Plasmodium falciparum malaria peptide correlates with protective immunity by Jindra Purmova; Luz Mary Salazar; Fabiola Espejo; Mary Helena Torres; Marcia Cubillos; Elizabeth Torres; Yolanda Lopez; Raul Rodrı́guez; Manuel Elkin Patarroyo (27-33).
Apical membrane antigen-1 is an integral Plasmodium falciparum malaria parasite membrane protein. High activity binding peptides (HABPs) to human red blood cells (RBCs) have been identified in this protein. One of them (peptide 4313), for which critical binding residues have already been defined, is conserved and nonimmunogenic. Its critical binding residues were changed for amino acids having similar mass but different charge to change such immunological properties; these changes generated peptide analogues. Some of these peptide analogues became immunogenic and protective in Aotus monkeys.Three-dimensional models of peptide 4313 and three analogues having different immune characteristics, were calculated from nuclear magnetic resonance (NMR) experiments with distance geometry and restrained molecular dynamic methods. All peptides contained a β-turn structure spanning amino acids 7 to 10, except randomly structured 4313. When analysing dihedral angle φ and ψ values, distorted type III or III′ turns were identified in the protective and/or immunogenic peptides, whilst classical type III turns were found for the nonimmunogenic nonprotective peptides. This data shows that some structural modifications may lead to induction of immunogenicity and/or protection, suggesting a new way to develop multicomponent, subunit-based malarial vaccines.
Keywords: Malaria; Peptide; Conformation; Nuclear magnetic resonance; Apical merozoite antigen-1 protein;

Cholesterol esterase accelerates intestinal cholesterol absorption by Ikuo Ikeda; Ryosuke Matsuoka; Tadateru Hamada; Kosuke Mitsui; Sachiko Imabayashi; Akira Uchino; Masao Sato; Eiichi Kuwano; Tomoaki Itamura; Koji Yamada; Kazunari Tanaka; Katsumi Imaizumi (34-44).
Mechanisms of acceleration of cholesterol absorption by cholesterol esterase were investigated in various experimental conditions. Lymphatic recovery of cholesterol intubated as a micellar solution containing phosphatidylcholine (PC) into the duodenum was enhanced by the co-administration of cholesterol esterase in rats drained of bile and pancreatic juice. However, no accelerated incorporation was observed when cholesterol was solubilized in PC-depleted micelles. Cholesterol esterase dose-dependently accelerated the incorporation of cholesterol into differentiated Caco-2 cells, only when cholesterol was solubilized in PC-containing micelles. The accelerated incorporation of cholesterol into Caco-2 cells by cholesterol esterase disappeared when the enzyme was preincubated with a suicide inhibitor of cholesterol esterase. Cholesterol esterase has an activity as phospholipase A2. When 10% of PC in bile salt micelles was replaced by lysophosphatidylcholine (lysoPC), the incorporation of cholesterol into Caco-2 cells was significantly accelerated. Cholesterol esterase enhanced the incorporation of micellar cholesterol into brush border membranes prepared from the rat jejunum. The addition of cholesterol esterase to bile salt micelles accelerated the release of micellar cholesterol in a dose-dependent manner, only when the micelles contained PC. These observations strongly suggest that cholesterol esterase hydrolyzes PC in bile salt micelles and thereby, accelerating the release of cholesterol from bile salt micelles. This may be a major cause of the acceleration of cholesterol absorption by cholesterol esterase.
Keywords: Cholesterol esterase; Caco-2 cells; Cholesterol absorption; Phosphatidylcholine; Phospholipase A2;

Formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) in solutions of free 2′-deoxyguanosine (dG) and calf thymus DNA (DNA) was compared for the diffusion-dependent and localised production of oxygen radicals from phosphate-mediated oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+). The oxidation of Fe2+ to Fe3+ was followed at 304 nm at pH 7.2 under aerobic conditions. Given that the concentration of Fe2+ ≥phosphate concentration, the rate of Fe2+ oxidation was significantly higher in DNA-phosphate as compared for the same concentration of inorganic phosphate. Phosphate catalysed oxidation of ferrous ions in solutions of dG or DNA led through the production of reactive oxygen species to the formation of 8-oxo-dG. The yield of 8-oxo-dG in solutions of dG or DNA correlated positively with the inorganic-/DNA-phosphate concentrations as well as with the concentrations of ferrous ions added. The yield of 8-oxo-dG per unit oxidised Fe2+ were similar for dG and DNA; thus, it differed markedly from radiation-induced 8-oxo-dG, where the yield in DNA was several fold higher.For DNA in solution, the localisation of the phosphate ferrous iron complex relative to the target is an important factor for the yield of 8-oxo-dG. This was supported from the observation that the yield of 8-oxo-dG in solutions of dG was significantly increased over that in DNA only when Fe2+ was oxidised in a high excess of inorganic phosphate (50 mM) and from the lower protection of DNA damage by the radical scavenger (hydroxymethyl)aminomethane (Tris)–HCl.
Keywords: 8-oxo-dG; Fenton; DNA; 2′-Deoxyguanosine; Phosphate; Iron;

The effects of water-soluble nonstarch polysaccharides (sNSP) on human metabolism are considered to be beneficial because they decrease postprandial glycaemia and insulinaemia following ingestion of starch-rich foods. The mechanisms by which sNSP attenuate the postprandial rise in blood glucose are not well understood but their presence increases the viscosity of gastrointestinal contents, which affects physiological functions, e.g. gastric emptying and peristalsis. Increased viscosity and decreased water activity during hydrothermal treatment of starch could influence α-amylase action.Using guar galactomannan as a representative of sNSP, we found that galactomannan has a direct noncompetitive inhibitory effect on α-amylase with a K i value of approximately 0.5% (3.3 μM). The inhibition is not time dependent and studies suggest direct binding of the enzyme to galactomannan; the resulting galactomannan–amylase complex being inactive. Processing of starch at low water levels greatly affects the catalytic efficiency of α-amylase. The K m value for starch heat treated in limited water is raised and k cat is lowered relative to starch gelatinised in excess water. Since galactomannan has no effect on the K m of α-amylase, we conclude that the inhibitory action of the polymer is not secondary to a decrease in available water. Neither does it seem to be a consequence of impaired diffusion of enzyme, substrate and products because of an increase in viscosity of the medium.Thus, the effects of sNSP in lowering postprandial glycaemia not only involve modifications of gut physiology, but also include direct inhibition of the first stage in the biochemical degradation of starch.
Keywords: α-Amylase; Nonstarch polysaccharide; Dietary fibre; Galactomannan inhibition; Hydrothermal treatment;

Isolation of lipoxygenase isoforms from Glycine max embryo axes based on apparent cross-reactivity with anti-myosin antibodies by Ignacio Islas-Flores; Salvador Corrales-Villamar; Elaine Bearer; Juan Carlos Raya; Marco A. Villanueva (64-70).
Three lipoxygenase isoforms were isolated from Glycine max embryo axes. A number of proteins around 97 kDa cross-reacted with several anti-actin and anti-myosin antibodies and these were used to follow their purification through gel filtration, hydroxyapatite and anion exchange columns. The 97-kDa cross-reactive material eluted in the unbound fractions of the last anion exchange column, and displayed two components of pI's 6.2 and 6.3. Further phase partition of this fraction in TX-114 yielded a hydrophobic 97 kDa protein. Additionally, a 95-kDa protein was retained and eluted from this last column. Partial peptide sequences indicated that the 95 kDa protein was soybean lipoxygenase-1, the first 97 kDa protein was lypoxygenase-3, and the hydrophobic 97 kDa protein was lipoxygenase-2. Several possible reasons for the cross-reactivity with the antibodies are discussed. To our knowledge, this is the first example of individual lipoxygenase isoforms isolated from soybean embryo axes.
Keywords: Embryo; Lipoxidase; Lipoxygenase; Purification; Seed; Glycine max;

The riboflavin (RF, Vitamin B2) and flavin adenine dinucleotide (FAD)-sensitized photooxidation of tryptophan (TrpH) and tyrosine (TyrOH) were studied by laser flash photolysis. TrpH and TyrOH quench triplet flavin sensitizers to produce reduced flavin radicals (FlH) and oxidized radicals of TrpH or TyrOH (Trp and TyrO). Although Trp and TyrO cannot be observed directly by the laser flash photolysis, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), as a probe, was added to the system to result in the formation of radical cations of TMPD (TMPD •+) via quenching of Trp and TyrO, which provides more definitive proof of electron transfer in the photosensitization process than only direct observation of reduced flavin radicals. Electron transfer from TrpH and TyrOH to oxidized radicals of riboflavin and FAD with similar rate constants to the triplet flavins was observed for the first time, which may be a new way of TrpH and TyrOH damage. These results may shed new light on future application of flavins in photodynamic therapy, and imply that flavins might be applied potentially to photosensitization of oxygen deficiency or under high-intensity pulsed laser irradiation.
Keywords: Flavin; Tryptophan; Tyrosine; Electron transfer; N,N,N′,N′-tetramethyl-p-phenylenediamine; Oxidized radical;

The oxidation of linoleic acid leads to the generation of several products with biological activity, including 13-oxooctadeca-9,11-dienoic acid (13-OXO), a bioactive 2,4-dienone that has been linked to cell differentiation. In the current work, the conjugation of 13-OXO by human glutathione transferases (GSTs) of the alpha (A1–1, A4–4), mu (M1–1, M2–2) and pi (the allelic variants P1–1/ile, and P1–1/val) classes, and a rat theta (rT2–2) class enzyme has been evaluated. The kinetics and stereoselectivity of the production of the 13-OXO-glutathione conjugate (13-OXO-SG) have been examined. In contrast to many xenobiotic substrates, the endogenous substrate 13-OXO does not exhibit an appreciable non-enzymatic rate of conjugation under physiological conditions. Therefore, the GST-catalyzed conjugation takes on greater significance as it provides the only realistic means for formation of 13-OXO-SG in most biological systems. Alpha class enzymes are most efficient at catalyzing the formation of 13-OXO-SG with k cat/K m values of 8.9 mM−1 s−1 for GST A1–1 and 2.14 mM−1 s−1 for GST A4–4. In comparison, enzymes from the mu and pi classes exhibit specificity constants from 0.4 to 0.8 mM−1 s−1. Conjugation of 13-OXO with glutathione at C-9 of the substrate can yield a pair of diastereomers that can be resolved by chiral HPLC. GSTs from the mu and pi classes are the most stereoselective enzymes and there is no apparent relationship between catalytic efficiency and stereoselectivity. The role of GST in the metabolic disposition of the bioactive oxidation products of linoleic acid has implications for the regulation of normal cellular functions by these versatile enzymes.
Keywords: Glutathione transferase; 13-oxooctadecadienoic acid; Lipid oxidation; Linoleic acid oxidation; Glutathione conjugation;