INSECT CHYMOTRYPSINS: CHLOROMETHYL KETONE INACTIVATION AND SUBSTRATE SPECIFICITY RELATIVE TO POSSIBLE COEVOLUTIONAL ADAPTATION OF INSECTS AND PLANTS

Insect digestive chymotrypsins are present in a large variety of insect orders but their substrate specificity still remains unclear. Ewer insect chymotrypsins from 3 different insect orders (Dictyoptera, Coleoptera and two Lepidoptera) were isolated using affinity chromatography. Enzymes presented molecular masses in the range of 20 to 31 kDa and pH optima in the range of 7.5 to 10.0. Kinetic characterization. using different, colorimetric and fluorescent substrates indicated that insect chymotrypsins differ from, bovine chymotrypsin in their primary specificity toward small substrates (like N-benzoyl-L-Tyr p-nitroanilide) rather than on their preference for large substrates (exemplified by Succynil-Ala-Ala-Pro-Phe P-nitroanilide). Chloromethyl ketones (TPCK, N-alpha-tosyl-L-Phe chloromethyl ketone and Z-GGF-CK, N-carbobenzoxy-Gly-Gly-phe-CK) inactivated all chymotrypsins legated. Inactivation rates follow apparent first-order kinetics with variable second order rates (TPCK, 42 to 130 M(-1)s(-1); Z-GGF-CK, 150 to 450 M(-1)s(-1) that may be remarkably low for S. frugiperda chymotrypsin (TPCK, 6 M(-1)s(-1); Z-GGF-CK, 6.1 M(-1) s(-1)). Homology modelling and sequence alignment showed that. in lepidopteran chymotrypsins, differences in the amino acid residues in the neighborhood of the catalytic His 57 may affect its pKa, value. This is Proposed as the cause of the decrease in His 57 reactivity toward chloromethyl ketones. Such amino acid replacement in the active site is proposed. to be an adaptation to the presence of dietary ketones. (C) 2009 Wiley Periodicals, Inc.

Subsite substrate specificity of midgut insect chymotrypsins

Insect chymotrypsins are distinctively sensitive to plant protein inhibitors, suggesting that they differ in subsite architecture and hence in substrate specificities. Purified digestive chymotrypsins from insects of three different orders were assayed with internally quenched fluorescent oligopeptides with three different amino acids at P1 (Tyr, Phe, and Leu) and 13 amino acid replacements in positions P1`, P2, and P3. The binding energy (Delta G(s), calculated from Km values) and the activation energy (Delta G(T)(double dagger), determined from k(cat)/K-m values) were calculated. The hydrophobicities of each subsite were calculated from the efficiency of hydrolysis of the different amino acid replacements at that subsite. The results showed that except for S1, the other subsites (S2, S3, and S1`) vary among chymotrypsins. This result contrasts with insect trypsin data that revealed a trend along evolution, putatively associated with resistance to plant inhibitors. In spite of those differences, the data suggested that in lepidopteran chymotrypsins S2 and S1` bind the substrate ground state, whereas only S1` binds the transition state, supporting aspects of the present accepted mechanism of catalysis. 2008 Elsevier Ltd. All rights reserved.

Mechanism of Trypanosoma cruzi death induced by Cratylia mollis seed lectin

Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca(2+) containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 A mu g/ml) induced plasma membrane permeabilization followed by Ca(2+) influx and mitochondrial Ca(2+) accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (Delta I(m)) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca(2+) treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased Delta I(m) by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigotes death by necrosis.

Protection of spruce seedlings against pine weevil attacks by treatment of seeds or seedlings with nicotinamide, nicotinic acid and jasmonic acid

Sustainable methods are required to protect newly planted tree seedlings from insect herbivore attack. To this end, here Norway spruce (Picea abies (L.) Karst.) seeds were treated with 2.5 mM nicotinamide (NIC), 2.5 mM nicotinic acid (NIA), 3 mM jasmonic acid (JA) or 0.2 mM 5-azacytidine (5-Aza), and 6-month-old seedlings grown from these seeds were planted at a reforestation area in central Sweden. Attack by pine weevils (Hylobius abietis) was reduced by 50 per cent by NIC treatment, 62.5 per cent by JA treatment and 25 per cent by 5-Aza treatment, when compared with seedlings grown from untreated seeds. Watering 18-month-old spruce seedlings with 2 mM NIC or 2 mM NIA did reduce attack during the first season in the field by 40 and 53 per cent, respectively, compared with untreated plants. Girdling was also reduced by the different treatments. Analysis of conifer seedlings treated with 5-Aza points at a possible involvement of epigenetic mechanisms in this defensive capacity. This is supported by a reduced level of DNA methylation in the needles of young spruce seedlings grown in a greenhouse from NIC-treated seeds. Seed treatment for seedling defense potentiation is simple, inexpensive and also a new approach for forestry with many potential applications.

The Predation of Artemia Nauplii by the Larvae of the Amazon River Prawn, Macrobrachium amazonicum (Heller, 1862), is Affected by Prey Density, Time of Day, and Ontogenetic Development

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A scanning electron microscopy study of the seed and post-seminal development in Angelonia salicariifolia Bonpl. (Scrophulariaceae)

The external morphology of seeds and post-germination developmental stages of Angelonia salicariifolia Bonpl. (Scrophulariaceae) were investigated using scanning electron microscopy. Some structural features of the seed exotesta and seedling in Angelonia are presented for the first time and are of potential taxonomic value for this neotropical genus. The seeds are very small (0.9-1.7 mm long and 0.5-0.9 mm wide), ovate, with a reticulate-crested exotesta, reticules arranged uniformly in longitudinal rows, with a high density of microcilia-like projections on the cell wall of the reticule base and on the edge of the crests. The hilum is located beside the micropyle at the narrow end of the seed. Germination is epigeal. During germination the radicle develops, followed by elongation of the hypocotyl and primary root. At this stage dense root hairs develop on the lower part of the hypocotyl. The apical bud-located between the cotyledons-begins to develop after the cotyledons have unfolded. The cotyledons are equal in size, sessile and ovate. The seedlings have two types of trichomes, one characteristic of the cotyledons and first pair of leaves (glandular, sessile, four-celled head with quadrangular shape) and the other characteristic of the hypocotyl and epicotyl (stalked, erect, elongate and three-celled with dome-shaped unicellular head). (C) 2001 Annals of Botany Company.

Efeito de genótipos de caupi, Vigna unguiculata (L.) Walp., sobre o desenvolvimento de Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Electrical conductivity of the seed soaking solution and soybean seedling emergence

Vigor of soybean [Glycine max (L.) Merrill] seeds can be evaluated by measuring the electrical conductivity (EC) of the seed soaking solution, which has shown a satisfactory relationship with field seedling emergence, but has not had aproper definition of range yet. This work studies the relationship between EC and soybean seedling emergence both in the field and laboratory conditions, using twenty two seed lots. Seed water content, standard germination and vigor (EC, accelerated aging and cold tests) were evaluated under laboratory conditions using -0.03; -0.20; -0.40 and -0.60 MPa matric potentials, and field seedling emergence was also observed. There was direct relationship between EC and field seedling emergence (FE). Under laboratory conditions, a decreasing relationship was found between EC and FE as water content in the substrate decreased, Relationships between these two parameters were also found when -0.03; -0.20 and -0.40 MPa matric potentials were used. EC tests can be used successfully to evaluate soybean seed vigor and identify lots with higher or lower field emergence potential.

Accelerated ageing test to evaluate seed vigor in pumpkin and zucchini seeds

The accelerated ageing (AA) test is widely used to evaluate seed vigor in various species including vegetable crops. The objective of this work was to study procedures for conducting AA to evaluate seed vigor of pumpkin and zucchini seed lots. Five seed lots of commercially acceptable quality (germination) standards were selected from both cv. `Menina Brasileira' (zucchini) and `Barbara' Hybrid (pumpkin). The following tests were conducted: standard germination, the first count of germination, and AA with and without saline solution (periods of 48, 72 and 96 hours). For each combination of temperature and ageing period, seeds were placed in a single layer on a screen in a germination plastic box with either 40 mL deionized water or 40g NaCl/100mL of water. Both AA methods identified lots 2 and 5 of `Menina Brasileira' and lot 10 of `Barbara' hybrid as having the lowest physiological quality. The temperature 41 C promoted a more drastic reduction in germination than did 38 C. This observation was more marked after 96 hours. Based on the results obtained, it can be concluded that the combination 41 degrees C/96 hours should be used to evaluate the physiological potential of pumpkin and zucchini seeds; however, additional studies are necessary to evaluate other combinations and confirm this indication.

The amount of solar energy made available for the production of a sabia (Mimosa caesalpinaefolia Benth.) seed has a determinating effect on its becoming dormant

The amount of solar energy made available for the production of a sabid seed varied as a function of the time of the year, the face of the plant in which, the position in the plant on which and the position in the pod in which it was produced.Variation in solar energy availability as a consequence of the time of the year was a direct consequence of latitude. At 21degrees5'22 S the highest amounts of Global Solar Radiation (GSR) reaching the site where the experiment was conducted took place during the months from November through February. During these months there were no marked differences between any two of the amounts of GSR reaching faces North (N), South (S), West (W) East (E). From February through November (period during which the sabid plants of this study flowered and the resulting seeds matured and were harvested) the total GSR's were the lowest and marked differences were found between faces N and S, with face N receiving much more GSR than face S. During that period, faces W and E received practically the same amount of GSR and it was much less than that received by face N and much more than the one received by face S.The amount of biological energy made available for the development of a seed seemed also to vary according to a dry matter partitioning strategy by the plant -the central third of the plant seemed to be the one receiving the highest amounts of energy, followed either by the upper or the lower third of the plant- it was not very clear which third of the plant immediately followed the central one. The partitioning of biological energy at the pod level also seemed to follow a strategy by which the central seeds would be the ones to receive more, followed by the proximal seeds and these by the distal ones.This availability of energy seemed to have a direct effect on seed size, weight and on the percentage of seeds which showed a degree of dormancy deep enough to prevent their germinating under the conditions of a standard germination test.The implications of these results for the improvement of methods for the overcoming of dormancy of sabia seeds are discussed.

Temperature during soybean seed storage and the amount of electrolytes of soaked seeds solution

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Seed-borne pathogens and electrical conductivity of soybean seeds

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Electrical conductivity as an indicator of pea seed aging of stored at different temperatures

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Relationship of black layer and milk line development on maize seed maturity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)