BBA - General Subjects (v.1571, #2)

Talisia esculenta lectin and larval development of Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) by Maria Ligia R. Macedo; Maria das Graças Machado Freire; José Camillo Novello; Sérgio Marangoni (83-88).
Bruchid larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil and the Mexican bean weevil, are pests that damage stored seeds. Plant lectins have been implicated as antibiosis factors against insects, particularly the cowpea weevil, Callosobruchus maculatus. Talisia esculenta lectin (TEL) was tested for anti-insect activity against C. maculatus and Zabrotes subfasciatus larvae. TEL produced ca. 90% mortality to these bruchids when incorporated in an artificial diet at a level of 2% (w/w). The LD50 and ED50 for TEL was ca. 1% (w/w) for both insects. TEL was not digested by midgut preparations of C. maculatus and Z. subfasciatus. The transformation of the genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.
Keywords: Talisia esculenta lectin; Callosobruchus maculatus; Zabrotes subfasciatus; Resistance;

First observation of solution structures of bradykinin–penta-O-galloyl-d-glucopyranose complexes as determined by NMR and simulated annealing by Sarah Vergé; Tristan Richard; Serge Moreau; Alain Nurich; Jean-Michel Merillon; Joseph Vercauteren; Jean-Pierre Monti (89-101).
Polyphenols (tannins) are known for their high propensity to precipitate proteins. They bind most strongly to proteins with a high proline content. Understanding the mechanism of this association is of prime interest because this interaction might induce protein conformational changes that may modify their biological activity. To investigate the interaction, an NMR study was carried out on the binding of a representative polyphenol, penta-O-galloyl-d-glucopyranose, to a nonapeptide hormone, bradykinin (BDK), where proline accounts for 30% of residues. Series of 1D and 2D-NMR experiments were performed. For the first time, a three-dimensional structure of complexes was determined using 2D-NMR experiments and molecular modeling. These structure calculations are a potent tool to understand how the association arises. They clearly show that the interaction is a complex phenomenon where several parameters are involved. The PGG/BDK complexes are formed by multiple weak interactions between peptide side chains and galloyl rings. Proline and arginine are good anchoring points and the glycine gives a certain flexibility in the peptide backbone that allows the polyphenol to approach and interact. Therefore, it is not only the hydrophobic stackings between galloyl rings and proline and hydrogen bonding involving arginine and aromatic rings which are important. The residue sequence and the side chain steric bulk also intervene.
Keywords: Bradikynin; Penta-O-galloyl-d-glucopyranose; NMR;

Mechanisms of flavonoid repair reactions with amino acid radicals in models of biological systems: a pulse radiolysis study in micelles and human serum albumin by Paulo Filipe; Patrice Morlière; Larry K. Patterson; Gordon L. Hug; Jean-Claude Mazière; Cécile Mazière; João P. Freitas; Afonso Fernandes; René Santus (102-114).
Neutral tryptophan (Trp) and tyrosine (TyrO) radicals are repaired by certain flavonoids in buffer, in micelles and in human serum albumin (HSA) with corresponding formation of semioxidized flavonoid radicals. In deaerated buffer, Trp but not TyrO radicals react with catechin. In micelles, quercetin and rutin repair both Trp and TyrO radicals. In addition to amino acid reactivity, microenvironmental factors and nature of the flavonoids govern this repair. Electron transfer efficiencies from quercetin to negatively charged Trp radicals are 100% in the micellar pseudophases of positively charged cetyltrimethylammonium bromide, (CTAB), and neutral Triton X100 (TX100), but 55% in negatively charged sodium dodecyl sulfate (SDS). In oxygen-saturated CTAB micelles, quercetin also reacts with the superoxide radical anion. When bound to domain IIA of HSA, quercetin repairs, by intra- or intermolecular encounter, less than 20% of oxidative damage to HSA. Quercetin can also repair freely circulating oxidized molecules with repair efficiencies falling to 7% for oxidized Trp, Tyr and α-MSH and to less than 2% for urate radical. This limited effectiveness is attributed both to the inaccessibility of bound quercetin and rutin toward radicals of circulating molecules and to the diffusion-controlled recombination of these radicals.
Keywords: Tryptophan; Tyrosine; Radical; α-MSH; Urate; Kinetics;

An aqueous acetone extract obtained from the pericarps of Mallotus japonicus (MJE) was observed to inhibit prostaglandin (PG) E2 production in a lipopolysaccharide (LPS)-activated murine macrophage-like cell line, RAW 264.7. Six phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against PGE2 production. Among these phloroglucinol derivatives, isomallotochromanol showed the strongest inhibitory activity, with an IC50 of 1.0 μM. MJE and its phloroglucinol derivatives did not effect the enzyme activity of either prostaglandin endoperoxide synthase (PGHS)-1 or PGHS-2. However, induction of PGHS-2 in LPS-activated macrophages was inhibited by MJE and its phloroglucinol derivatives, whereas the level of PGHS-1 protein was not affected. Moreover, RT-PCR analysis showed that MJE and its phloroglucinol derivatives significantly suppressed PGHS-2 mRNA expression. Therefore, the observed inhibition of PGHS-2 induction by MJE and its phloroglucinol derivatives was likely due to a suppression of PGHS-2 mRNA expression. These results suggest that MJE and its phloroglucinol derivatives have the pharmacological ability to suppress PGE2 production by activated macrophages.
Keywords: Mallotus japonicus; Euphorbiaceae; Phloroglucinol derivative; Macrophage; Prostaglandin E2; Prostaglandin endoperoxide synthase;

Generation of carbon monoxide and iron from hemeproteins in the presence of 7,8-dihydroneopterin by Renate Horejsi; Christiane Jung; Reinhard Möller; Erwin Tafeit; Gilbert Reibnegger (124-130).
7,8-Dihydroneopterin and neopterin are secreted by human and primate macrophages after activation by interferon-γ in a ratio of 2:1. 7,8-Dihydroneopterin is known to suppress radical-mediated processes, but it is also able in the presence of iron ions to generate superoxide radical anion and hydroxyl radicals from molecular oxygen. Effects of 7,8-dihydroneopterin were investigated on (met)myoglobin and (met)hemoglobin. Addition of 7,8-dihydroneopterin to heme proteins in air-saturated solution resulted in dose-dependent cleavage of the porphyrin moiety. The liberation of non-heme iron and carbon monoxide originating from the cleaved porphyrin was quantified. Both were generated at equimolar concentrations with a linear correlation coefficient of 0.9. Addition of ferrous iron significantly accelerated the pteridine-mediated cleaving of the porphyrin. However, the total yield of porphyrin cleaved was controlled by the pterin rather than by the ferrous ion concentration. 7,8-Dihydroneopterin is assumed to reduce the heme iron in intact protein molecules, thereby preparing the conditions for binding of oxygen and carbon monoxide as ligands. Beyond that, it is concluded that hydroxyl radicals might be generated via reduction of molecular oxygen to superoxide anion in the autoxidation process and dismutation to hydrogen peroxide and subsequent Fenton reaction.
Keywords: 7,8-Dihydroneopterin; Hemoglobin; Myoglobin; Porphyrin cleavage;

Regulation of glutamine metabolism during the development of Bombyx mori larvae by Chikara Hirayama; Masatoshi Nakamura (131-137).
Waste ammonia is re-assimilated into amino acids via the amide group of glutamine and the amino group of glutamate (i.e. through glutamine synthetase/glutamate synthase pathway) for silk synthesis in the silkworm, Bombyx mori, in the last larval stadium. Glutamine concentration in hemolymph gradually decreased with the progress of the fifth instar and it remained at very low levels during the spinning stage, then followed by a sharp increase at the larval–pupal ecdysis. The changes in glutamine synthetase (GS) activity in silkworm tissues were relatively small through the larval development, while the changes in glutamate synthase (GOGAT) activity, especially in the posterior silk glands, were more drastic. In addition, activities of GOGAT in the tissues were much higher than those of the other enzymes involved in glutamine utilization, suggesting that glutamine pool was regulated mainly by the changes in GOGAT activity. Western blot analysis indicated that the changes in GOGAT protein level correlated with the changes in GOGAT activity. Topical application of a juvenile hormone analogue, methoprene, induced an accumulation of glutamine in the hemolymph of the fifth instar larvae. The levels of GOGAT protein and activity in the tissues of the methoprene treated larvae were much lower than those of the control larvae, whereas the methoprene treatment had no effect on the levels of GS activity. In conclusion, GOGAT expression promoted by reduction of juvenile hormone titer is quite important for enhanced utilization of nitrogen for synthesis of silk protein during the last larval instar.
Keywords: Glutamine synthetase (GS); Glutamate synthase (GOGAT); Glutamine; Silkworm (Bombyx mori); Silk synthesis; Juvenile hormone;

Induction of gluconeogenesis is accelerated in larvae of the insect Manduca sexta L. parasitized by Cotesia congregata (Say), maintaining the concentration of the blood sugar trehalose, an important nutrient for parasite development. Investigation has demonstrated that when host larvae are offered a choice of diets with varying levels of sucrose and casein, parasitized insects consume a different balance of these nutrients, principally due to a decrease in protein consumption. The result is metabolic homeostasis, with normal unparasitized and parasitized larvae exhibiting similar levels of gluconeogenesis and blood sugar level. In the present study, normal unparasitized and parasitized larvae were maintained on individual chemically defined diets having the balance of protein and carbohydrate consumed by each when offered a dietary choice. Total dietary nutrient, the sum of carbohydrate and protein, was provided at six levels, composed of three pairs of diets. Each diet pair consisting of diets having equivalent overall nutrient ratios of 2:1 and 1:1 casein/sucrose. Host growth and diet consumption were significantly affected by dietary nutrient level and the magnitude of these effects was influenced by parasitism. Due to the effects of dietary nutrient level on diet consumption, none of the unparasitized and parasitized larvae within any of the three diet pairs consumed protein and carbohydrate at the levels predicted by the earlier choice experiments. Among insects on all of the diets, however, two groups of unparasitized and parasitized larvae consumed the expected levels of protein and carbohydrate. In each case, gluconeogenesis, as measured by 13C nuclear magnetic resonance spectroscopy (NMR) analysis of pyruvate cycling and trehalose synthesis from [2-13C]pyruvate, was evident in unparasitized and parasitized insects, confirming the conclusions of the earlier experiments. Generally, all larvae that consumed less than approximately 250 mg of sucrose over the 3-day feeding period, were gluconeogenic, regardless of diet. Differential carbohydrate consumption, therefore, was an important factor in inducing gluconeogenesis in both unparasitized and parasitized insects. The selective 13C enrichment in trehalose displayed by non-gluconeogenic larvae on some diets demonstrated trehalose formation from [2]pyruvate. The absence of net carbohydrate synthesis in these insects was likely due to an elevation of glycolysis. There was no significant effect of diet consumption or parasitism on blood trehalose level. Parasitized larvae displayed higher levels of gluconeogenesis than did unparasitized insects, a finding consistent with the conclusion that blood sugar is rapidly sequestered by developing parasites. The parasite burden, the total number of parasites developing within host larvae, as well as the number of parasites emerging from host larvae to complete development, was significantly less at the lowest dietary nutrient level, but was otherwise similar at all dietary nutrient levels. Moreover, the number of parasites that emerged increased with increasing diet consumption as reflected by host final weight.
Keywords: Gluconeogenesis; Nutrition; 13C NMR; Parasitism; Pyruvate cycling; Manduca sexta; Cotesia congregata;

Previously, my colleagues and I have reported that the immunopurified hyper-phosphorylated retinoblastoma protein (ppRb) stimulates the activity of DNA polymerase α. I describe here the biochemical characteristics of this stimulatory activity. DNA polymerase α-stimulatory activity of ppRb was most remarkable when using activated DNA as a template-primer, rather than using poly(dT)-(rA)10, poly(dA)-(dT)12–18, and so on. Kinetic analysis showed that there was no significant difference in K m value for deoxyribonucleotides of DNA polymerase α in the presence of ppRb. Adding ppRb resulted in the overcoming pause site on the template, but did not affect the rate of misincorporation of incorrect deoxyribonucleotides. By adding ppRb, the optimal concentration of template-primer was shifted to a higher region, but not using M13 singly primed DNA. The ppRb seemed to assist the process that DNA polymerase α changed its conformation resulting in appropriate enzyme activity. These results suggest that ppRb affects both template-primer and DNA polymerase α and makes appropriate circumstances for the enzyme reaction.
Keywords: Retinoblastoma protein; DNA polymerase α; Stimulatory factor; Kinetics;

In the presence of iron or copper ions, the course of the oxidation in air of 5-S-cysteinyldopa (1), the main biosynthetic precursor of pheomelanins and trichochromes, was markedly changed affording two main products. One of these was identified as the oxobenzothiazine 8, previously obtained under nonphysiologically relevant conditions, while the other was characterized as the novel hydroxybenzothiazole 9. Besides 8 and 9, carboxylated and noncarboxylated benzothiazine products were obtained by persulfate oxidation of 1 in the presence of iron or copper ions. The ratio of formation yields of carboxylated/noncarboxylated benzothiazines, determined after reduction of the mixture, was lower than that of the control reaction run in the absence of metal ions, and much lower than that of the oxidation carried out in the presence of zinc ions, in agreement with a recent report. Notably, 8 and 9 were formed in variable yields under different oxidation conditions including tyrosinase/O2, peroxidase/hydrogen peroxide, and the hydrogen peroxide/or (9E,11Z,13S)-13-hydroperoxyoctadeca-9,11-dienoic acid/Fe(III) systems. Mechanistic routes to 8 and 9 were proposed based on the results of experiments involving in situ generation of labile benzothiazine intermediates. Overall, these results allow to formulate an improved biosynthetic scheme in which metal ions act as critical regulatory factors determining pheomelanin vs. trichochromes formation.
Keywords: Metal ion; 5-S-cysteinyldopa oxidation; Benzothiazine ring contraction; Benzothiazole intermediate; Melanogenesis regulation;