Chemical Modifications of Vicilins Interfere with Chitin-Binding Affinity and Toxicity to Callosobruchus maculatus (Coleoptera: Chrysomelidae) Insect: A Combined In Vitro and In Silico Analysis.

Vicilins are related to cowpea seed resistance toward Callosobruchus maculatus due to their ability to bind to chitinous structures lining larval midgut. However, this binding mechanism is not fully understood. Here, we identified chitin binding sites and investigated how in vitro and in silico chemical modifications interfere with vicilin chitin binding and insect toxicity. In vitro assays showed that unmodified vicilin strongly binds to chitin matrices, mainly with acetylated chitin. Chemical modifications of specific amino acids (tryptophan, lysine, tyrosine), as well as glutaraldehyde cross-linking, decreased the evaluated parameters. In silico analyses identified at least one chitin binding site in vicilin monomer, the region between Arg208 and Lys216, which bears the sequence REGIRELMK and forms an α helix, exposed in the 3D structure. In silico modifications of Lys223 (acetylated at its terminal nitrogen) and Trp316 (iodinated to 7-iodine-L-tryptophan or oxidized to ß-oxy-indolylalanine) decreased vicilin chitin binding affinity. Glucose, sucrose, and N-acetylglucosamine also interfered with vicilin chitin binding affinity.

Characterization and antifungal activity of a plant peptide expressed in the interaction between Capsicum annuum fruits and the anthracnose fungus.

Plant defensins are low molecular weight basic peptides ranging from 5 to 7 kDa, with capacity of inhibiting various pathogens, including fungi. They are present in different tissues of plants, including floral parts and fruits of Capsicum sp. The IIF48 extract, present in immature fruits of Capsicum annuum inoculated with C. gloeosporioides, was able to inhibit up to 100% growth 'in vitro' of the fungus Colletotrichum gloeosporioides. The main objective of this work was the purification and antifungal activity characterization of a defense-related plant defensin-like isolated of the IIF48 immature fruits extract. The IIF48 extract was subjected to HPLC purification and 13 fractions were obtained, followed by a tricine gel electrophoresis to obtain the protein profile. The different fractions were submitted to a growth inhibition assay against C. gloeosporioides fungus. Fraction 7 (F7) was the most active causing 73% inhibition. Because of the higher F7 activity and the presence of only a peptide of approximately 5 kDa this fraction was subjected to N-terminal sequencing. F7 fraction was carried out plasma membrane permeabilization assays, induction of intracellular ROS production analysis and investigated mitochondrial membrane potential. The F7 fraction showed significant inhibitory activity on the tested fungus, besides promoting membrane permeabilization, induction of endogenous ROS production in Colletotrichum cells and impairing mitochondrial functionality. The first 18 amino acid sequence of the F7 fraction peptide suggests homology to plant-like defensin and was named IIFF7Ca. We also concluded that IIFF7Ca peptide has an effective antimicrobial action against the fungus C. gloeosporioides.

A synthetic peptide derived of the ß2-ß3 loop of the plant defensin from Vigna unguiculata seeds induces Leishmania amazonensis apoptosis-like cell death.

For medical use of proteins and peptide-based drugs, it is desirable to have small biologically active sequences because they improve stability, reduce side effects, and production costs. Several plant defensins have their biological activities imparted by a sequence named γ-core. Vu-Def, a Vigna unguiculata defensin, has activity against Leishmania amazonensis, which is one etiological agent of leishmaniasis and for which new drugs are needed. Our intention was to understand if the region comprising the Vu-Def γ-core is responsible for the biological activity against L. amazonensis and to unveil its mechanism of action. Different microbiological assays with L. amazonensis in the presence of the synthetic peptide A36,42,44γ32-46Vu-Def were done, as well as ultrastructural and fluorescent analyses. A36,42,44γ32-46Vu-Def showed biological activity similar to Vu-Def. A36,42,44γ32-46Vu-Def (74 µM) caused 97% inhibition of L. amazonensis culture and parasites were unable to regrow in fresh medium. The cells of the treated parasites showed morphological alterations by ultrastructural analysis and fluorescent labelings that corroborate with the data of the organelles alterations. The general significance of our work is based on the description of a small synthetic peptide, A36,42,44γ32-46Vu-Def, which has activity on L. amazonensis and that the interaction between A36,42,44γ32-46Vu-Def-L. amazonensis results in parasite inhibition by the activation of an apoptotic-like cell death pathway.

Programmed cell death in soybean seed coats.

Seed coat is the tissue which establishes an interface between the seed inner tissues and external environment. Our group has shown that cowpea seed coat undergoes coordinated events of programmed cell death (PCD) during development. In relation to germinating seed coats, little is known on PCD events. The goal here was to investigate the biochemical aspects of germinating soybean seed coat, focusing on proteolytic activities related to PCD. In gel and in solution activity profiles of quiescent and germinating seed coat extracts revealed a complex pattern of caspase- and metacaspase-like cysteine protease activities. Trypsin inhibitor and reserve proteins were revealed as potential substrates for these proteases. A pancaspase inhibitor (z-VAD-CHO) affected the radicle length of seeds germinated under its presence. Ultrastructural analysis showed the absence of cell organelles in all seed coat layers after imbibition, while oligonucleosome fragments peaked at 72 h after imbibition (HAI). Altogether, the data suggest the presence of biochemical PCD hallmarks in germinating soybean seed coat and point to the involvement of the detected protease activities in processes such as reserve protein mobilization and weakening of seed coat.

Insight into the α-Amylase Inhibitory Activity of Plant Lipid Transfer Proteins.

We previously characterized the inhibitory activity of human salivary α-amylase (HSA) and Callosobruchus maculatus intestinal α-amylases by the plant lipid transfer protein from Vigna unguiculata ( Vu-LTP). Herein, we further study this inhibitory activity. First by an analysis of protein α-amylase inhibitors complexed with α-amylase, we find that positively charged amino acids of inhibitors interact with the active site of α-amylases and we know that Vu-LTP is rich in positively charged amino acid residues. For this reason, we model Vu-LTP, and based on its three-dimensional structure, we choose five peptides to be synthesized. Herein, we report that two peptides of Vu-LTP are responsible for HSA inhibition. A comparison of primary and tertiary structures of LTPs with and without inhibitory activity against α-amylase, superimposed with the sequence of Vu-LTP mapped for HSA inhibition, reinforces our suggestion that positively charged amino acids in loops are responsible for the inhibition. To prove our observation, one modified peptide is synthesized in which Arg39 is replaced by Gln. This modified peptide loses the HSA inhibitory property presented by the unmodified peptide. Therefore, we describe a new biological active for Vu-LTP, i.e. the α-amylase inhibitory activity that is not a fortuitous biological activity and probably has evolved to perform a biological function which is still unknown. A good candidate should be defense against insects. The results of this study also expand the possible biotechnological applications of LTPs.

A modified, hypoallergenic variant of the Ricinus communis Ric c1 protein retains biological activity.

Ric c1, an allergenic protein from Ricinus communis , is an insect α-amylase inhibitor that has become an occupational allergen. Ric c1 can cross-react with allergens from wheat, soybean, peanut, shrimp, fish, gluten, house dust, tobacco, and air fungus, thereby amplifying the concern and risks caused by Ricinus allergens. Two continuous IgE-binding epitopes were identified in Ric c1, both containing glutamic acid residues involved in IgE-binding and allergic challenges. We produced recombinant Ric c1 (rRic c1) in Escherichia coli , using primers from foliar R. communis DNA, and a mutant (Glu-Leu) recombinant protein (mrRic c1) in the same system using synthetic genes. rRic c1 preserved both allergenic and α-amylase inhibitory properties, and mrRic c1 drastically reduced allergenic properties. These results can help to establish meaningful relationships between structure, defense and allergenicity, important steps for producing engineered plants and developing new approaches for immunotherapy.

Purification and characterization of peptides from Capsicum annuum fruits which are α-amylase inhibitors and exhibit high antimicrobial activity against fungi of agronomic importance.

Proteins extracted from Capsicum annuum L. fruits were initially subjected to reversed-phase chromatography on HPLC, resulting in eight peptide-rich fractions. All the fractions obtained were tested for their ability to inhibit porcine trypsin and amylase from both human saliva and from larval insect in vitro. All fractions were also tested for their ability to inhibit growth of the phytopathogenic fungi. Several fractions inhibited the activity of human salivary amylase and larval insect amylase, especially fraction Fa5. No fraction tested was found to inhibit trypsin activity, being Fa2 fraction an exception. Interestingly fraction Fa5 also displayed high antimicrobial activity against the species of the Fusarium genus. Fraction Fa5 was found to have two major protein bands of 17 and 6.5 kDa, and these were sequenced by mass spectrometry. Two peptides were obtained from the 6.5-kDa band, which showed similarity to antimicrobial peptides. Fraction Fa5 was also tested for its ability to permeabilize membranes and induce ROS. Fraction Fa5 was able to permeabilize the membranes of all the fungi tested. Fungi belonging to the genus Fusarium also showed an increase in the endogenous production of ROS when treated with this fraction. Antimicrobial peptides were also identified in the fruits from other Capsicum species.

Identification of IgE-binding peptide and critical amino acids of Jatropha curcas allergen involved in allergenic response.

Increasing energy demand has spurred interest in the use of biofuels. Jatropha curcas (physic nut), an inedible oilseed, is a potential source of bioenergy. The seeds, however, contain allergens such as Jat c 1, a 2S albumin that can induce hypersensitivity reactions in humans and result in allergic diseases. Recent advances in identifying and characterizing plant allergens and, in particular, their immunoglobulin E (IgE)-binding epitopes have produced a wealth of information. We identified IgE-binding regions and the critical amino acids involved in the degranulation of mast cells and the release of histamine, preliminary steps for the prevention and treatment of this allergy. Four IgE-binding regions were identified in the sequence of Jat c 1. We identified and demonstrated the fundamental role of two glutamic acid residues in IgE binding. The sequence LEKQLEEGEVGS produces a random loop on the most exposed part of Jat c 1. This region is important to the stimulation of the allergic response. The possibility of using this information to produce vaccines and other pharmacological agents for allergy treatment is discussed.

Isolation, characterization and mechanism of action of an antimicrobial peptide from Lecythis pisonis seeds with inhibitory activity against Candida albicans.

Antimicrobial peptides (AMPs) are produced by a range of organisms as a first line of defense against invaders or competitors. Owing to their broad antimicrobial activity, AMPs have attracted attention as a potential source of chemotherapeutic drugs. The increasing prevalence of infections caused by Candida species as opportunistic pathogens in immunocompromised patients requires new drugs. Lecythis pisonis is a Lecythydaceae tree that grows in Brazil. The AMPs produced by this tree have not been described previously. We describe the isolation of 12 fractions enriched in peptides from L. pisonis seeds. Of the 12 fractions, at 10 µg/ml, the F4 fraction had the strongest growth inhibitory effect (53.7%) in Candida albicans, in addition to a loss of viability of 94.9%. The F4 fraction was separated into seven sub-fractions by reversed-phase chromatography. The F4.7' fraction had the strongest activity at 10 µg/ml, inhibiting C. albicans growth by 38.5% and a 69.3% loss of viability. The peptide in F4.7' was sequenced and was found to be similar to plant defensins. For this reason, the peptide was named L. pisonis defensin 1 (Lp-Def1). The mechanism of action that is responsible for C. albicans inhibition by Lp-Def1 includes a slight increase of reactive oxygen species induction and a significant loss of mitochondrial function. The results described here support the future development of plant defensins, specifically Lp-Def1, as new therapeutic substances against fungi, especially C. albicans.

Defense response in non-genomic model species: methyl jasmonate exposure reveals the passion fruit leaves' ability to assemble a cocktail of functionally diversified Kunitz-type trypsin inhibitors and recruit two of them against papain.

MAIN CONCLUSION: Multiplicity of protease inhibitors induced by predators may increase the understanding of a plant's intelligent behavior toward environmental challenges. Information about defense mechanisms of non-genomic model plant passion fruit (Passiflora edulis Sims) in response to predator attack is still limited. Here, via biochemical approaches, we showed its flexibility to build-up a broad repertoire of potent Kunitz-type trypsin inhibitors (KTIs) in response to methyl jasmonate. Seven inhibitors (20-25 kDa) were purified from exposed leaves by chromatographic techniques. Interestingly, the KTIs possessed truncated Kunitz motif in their N-terminus and some of them also presented non-consensus residues. Gelatin-Native-PAGE established multiple isoforms for each inhibitor. Significant differences regarding inhibitors' activity toward trypsin and chymotrypsin were observed, indicating functional polymorphism. Despite its rarity, two of them also inhibited papain, and such bifunctionality suggests a recruiting process onto another mechanistic class of target protease (cysteine-type). All inhibitors acted strongly on midgut proteases from sugarcane borer, Diatraea saccharalis (a lepidopteran insect) while in vivo assays supported their insecticide properties. Moreover, the bifunctional inhibitors displayed activity toward midgut proteases from cowpea weevil, Callosobruchus maculatus (a coleopteran insect). Unexpectedly, all inhibitors were highly effective against midgut proteases from Aedes aegypti a dipteran insect (vector of neglected tropical diseases) opening new avenues for plant-derived PIs for vector control-oriented research. Our results reflect the KTIs' complexities in passion fruit which could be wisely exploited by influencing plant defense conditions. Therefore, the potential of passion fruit as source of bioactive compounds with diversified biotechnological application was strengthened.

Functional expression and activity of the recombinant antifungal defensin PvD1r from Phaseolus vulgaris L. (common bean) seeds.

BACKGROUND: Defensins are basic, cysteine-rich antimicrobial peptides that are important components of plant defense against pathogens. Previously, we isolated a defensin, PvD1, from Phaseolus vulgaris L. (common bean) seeds. RESULTS: The aim of this study was to overexpress PvD1 in a prokaryotic system, verify the biologic function of recombinant PvD1 (PvD1r) by comparing the antimicrobial activity of PvD1r to that of the natural defensin, PvD1, and use a mutant Candida albicans strain that lacks the gene for sphingolipid biosynthesis to unravel the target site of the PvD1r in C. albicans cells. The cDNA encoding PvD1, which was previously obtained, was cloned into the pET-32 EK/LIC vector, and the resulting construct was used to transform bacterial cells (Rosetta Gami 2 (DE3) pLysS) leading to recombinant protein expression. After expression had been induced, PvD1r was purified, cleaved with enterokinase and repurified by chromatographic steps. N-terminal amino acid sequencing showed that the overall process of the recombinant production of PvD1r, including cleavage with the enterokinase, was successful. Additionally, modeling revealed that PvD1r had a structure that was similar to the defensin isolated from plants. Purified PvD1 and PvD1r possessed inhibitory activity against the growth of the wild-type pathogenic yeast strain C. albicans. Both defensins, however, did not present inhibitory activity against the mutant strain of C. albicans. Antifungal assays with the wild-type C. albicans strains showed morphological changes upon observation by light microscopy following growth assays. PvD1r was coupled to FITC, and the subsequent treatment of wild type C. albicans with DAPI revealed that the labeled peptide was intracellularly localized. In the mutant strain, no intracellular labeling was detected. CONCLUSION: Our results indicate that PvD1r retains full biological activity after recombinant production, enterokinase cleavage and purification. Additionally, our results from the antimicrobial assay, the microscopic analysis and the PvD1r-FITC labeling assays corroborate each other and lead us to suggest that the target of PvD1 in C. albicans cells is the sphingolipid glucosylceramide.

Thionin-like peptides from Capsicum annuum fruits with high activity against human pathogenic bacteria and yeasts.

Plants defend themselves against pathogens with production of antimicrobial peptides (AMPs). Herein we describe the discovery of a new antifungal and antibacterial peptide from fruits of Capsicum annuum that showed similarity to an already well characterized family of plant AMPs, thionins. Other fraction composed of two peptides, in which the major peptide also showed similarity to thionins. Among the obtained fractions, fraction 1, which is composed of a single peptide of 7 kDa, was sequenced by Edman method and its comparative sequence analysis in database (nr) showed similarity to thionin-like peptides. Tests against microorganisms, fraction 1 presented inhibitory activity to the cells of yeast Saccharomyces cerevisiae, Candida albicans, and Candida tropicalis and caused growth reduction to the bacteria species Escherichia coli and Pseudomonas aeruginosa. Fraction 3 caused inhibitory activity only for C. albicans and C. tropicalis. This fraction was composed of two peptides of ∼7 and 10 kDa, and the main protein band correspondent to the 7 kDa peptide, also showed similarity to thionins. This plasma membrane permeabilization assay demonstrates that the peptides present in the fractions 1 and 3 induced changes in the membranes of all yeast strains, leading to their permeabilization. Fraction 1 was capable of inhibiting acidification of the medium of glucose-induced S. cerevisiae cells 78% after an incubation time of 30 min, and opposite result was obtained for C. albicans. Experiments demonstrate that the fraction 1 and 3 were toxic and induced changes in the membranes of all yeast strains, leading to their permeabilization.

Activity of recombinant and natural defensins from Vigna unguiculata seeds against Leishmania amazonensis.

Antimicrobial peptides (AMPs), which are differentiated from other antibiotic peptides, such as gramicidins and polymyxins, because they are synthesized by large enzymatic complex and bear modified amino acids including d-amino acids, are short polymers of l-amino acids synthesized by ribosomes upon which all living organisms rely to defend themselves from invaders or competitor microorganisms. AMPs have received a great deal of attention from the scientific community as potential new drugs for neglected diseases such as Leishmaniasis. In plants, they include several families of compounds, including the plant defensins. The aim of the present study was to improve the expression of recombinant defensin from Vigna unguiculata seeds (Vu-Defr) and to test its activity against Leishmania amazonensis promatigotes. Recombinant expression was performed in LB and TB media and under different conditions. The purification of Vu-Defr was achieved by immobilized metal ion affinity and reversed-phase chromatography. The purified Vu-Defr was analyzed by circular dichroism (CD), and its biological activity was tested against L. amazonenis promastigotes. To demonstrate that the recombinant production of Vu-Defr did not interfere with its fold and biological activity, the results of all experiments were compared with the results from the natural defensin (Vu-Def). The CD spectra of both peptides presented good superimposition indicating that both peptides present very similar secondary structure and that the Vu-Defr was correctly folded. L. amazonensis treated with Vu-Defr led to the elimination of 54.3% and 46.9% of the parasites at 24 and 48h of incubation time, respectively. Vu-Def eliminated 50% and 54.8% of the parasites at 24 and 48 h, respectively. Both were used at a concentration of 100 µg/mL. These results suggested the potential for plant defensins to be used as new antiparasitic substances.

Síndrome diencéfalico como causa de desnutrición severa / Diencephalic syndrome as a cause of severe malnutrition

El síndrome diencefálico es un complejo de síntomas y signos causados por disfunción de esta área del encéfalo caracterizado por una marcada desnutrición aun cuando la ingesta calórica es normal. Se presentan dos casos, el primero de ellos una niña de 13 meses de edad con antecedentes de un fallo de medro a partir del tercer mes de vida, que ingresó en este servicio para el estudio de una desnutrición proteico energética severa que presentó en el transcurso de su evolución un apetito inestable y al mes de ingresada un evento paroxístico. Se le realizó resonancia magnética nuclear y se comprobó imagen hipodensa, redondeada, que medía aproximadamente 3 x 3 cm en región supraselar; fue intervenida quirúrgicamente en 2 ocasiones, se realizó exéresis del tumor, y se confirmó anatomopatológicamente un astrocitoma pilocítico de bajo grado. El segundo paciente, un lactante que ingresó con el diagnóstico confirmado de tumor intracraneal para estudio, semejante al caso presentado anteriormente, mostraba una marcada desnutrición proteico energética, se le realizó tomografía axial computarizada en la que se pudo apreciar una extensa masa tumoral supraselar con dilatación del sistema ventricular. Durante su evolución presentó marcada anorexia con pérdida de peso progresiva, por lo que se realizó gastrostomía. A los 59 días falleció como consecuencia de una pancitopenia, y la necropsia concluyó: astrocitoma pilocítico de bajo grado.

Diencephalic syndrome is a set of symptoms and signs caused by dysfunction in this area of the encephalon and characterized by marked malnutrition despite adequate intake of calories. Two cases were reported in this paper. The first one was a 13-years old girl with a history of medro failure since her 3rd month of life, who was admitted to this service for the study of her severe protein/energy malnutrition. In the course of her hospitalization, she presented with unstable appetite and suffered a paroxistic event one month after being hospitalized. The infant girl was then performed magnetic resonance imaging test which revealed a 3 cm x 3cm rounded hypodense image in the suprasellar region. She was operated on twice, the tumor was excised and the anatomic and pathological analysis yielded the presence of low grade pilocytic astrocytoma. The second patient was an small infant who was admitted to the service with a confirmed diagnosis of intracranial tumor for study, very similar to the case previously presented. He suffered an acute protein/energy malnutrition, so he was performed a computerized tomography which showed a broad tumoral mass in the suprasellar region with dilated ventricular system. During the hospitalization, he presented with marked anorexia, progressive loss of weight and he finally underwent gastrotomy, but he died 59 days after admission as a result of pancytopenia. The result of necropsy was low grade pilocytic astrocytoma.

Síndrome diencéfalico como causa de desnutrición severa / Diencephalic syndrome as a cause of severe malnutrition

El síndrome diencefálico es un complejo de síntomas y signos causados por disfunción de esta área del encéfalo caracterizado por una marcada desnutrición aun cuando la ingesta calórica es normal. Se presentan dos casos, el primero de ellos una niña de 13 meses de edad con antecedentes de un fallo de medro a partir del tercer mes de vida, que ingresó en este servicio para el estudio de una desnutrición proteico energética severa que presentó en el transcurso de su evolución un apetito inestable y al mes de ingresada un evento paroxístico. Se le realizó resonancia magnética nuclear y se comprobó imagen hipodensa, redondeada, que medía aproximadamente 3 x 3 cm en región supraselar; fue intervenida quirúrgicamente en 2 ocasiones, se realizó exéresis del tumor, y se confirmó anatomopatológicamente un astrocitoma pilocítico de bajo grado. El segundo paciente, un lactante que ingresó con el diagnóstico confirmado de tumor intracraneal para estudio, semejante al caso presentado anteriormente, mostraba una marcada desnutrición proteico energética, se le realizó tomografía axial computarizada en la que se pudo apreciar una extensa masa tumoral supraselar con dilatación del sistema ventricular. Durante su evolución presentó marcada anorexia con pérdida de peso progresiva, por lo que se realizó gastrostomía. A los 59 días falleció como consecuencia de una pancitopenia, y la necropsia concluyó: astrocitoma pilocítico de bajo grado(AU)

Diencephalic syndrome is a set of symptoms and signs caused by dysfunction in this area of the encephalon and characterized by marked malnutrition despite adequate intake of calories. Two cases were reported in this paper. The first one was a 13-years old girl with a history of medro failure since her 3rd month of life, who was admitted to this service for the study of her severe protein/energy malnutrition. In the course of her hospitalization, she presented with unstable appetite and suffered a paroxistic event one month after being hospitalized. The infant girl was then performed magnetic resonance imaging test which revealed a 3 cm x 3cm rounded hypodense image in the suprasellar region. She was operated on twice, the tumor was excised and the anatomic and pathological analysis yielded the presence of low grade pilocytic astrocytoma. The second patient was an small infant who was admitted to the service with a confirmed diagnosis of intracranial tumor for study, very similar to the case previously presented. He suffered an acute protein/energy malnutrition, so he was performed a computerized tomography which showed a broad tumoral mass in the suprasellar region with dilated ventricular system. During the hospitalization, he presented with marked anorexia, progressive loss of weight and he finally underwent gastrotomy, but he died 59 days after admission as a result of pancytopenia. The result of necropsy was low grade pilocytic astrocytoma(AU)

Purification of a Kunitz-type inhibitor from Acacia polyphyllaDC seeds: characterization and insecticidal properties against Anagasta kuehniella Zeller (Lepidoptera: Pyralidae).

Anagasta kuehniella is a polyphagous pest that causes economic losses worldwide. This species produces serine proteases as its major enzymes for protein digestion. In this study, a new serine-protease inhibitor was isolated from Acacia polyphylla seeds (AcKI).Further analysis revealed that AcKI is formed by two polypeptide chains with a relative molecular mass of ∼20 kDa. The effects of AcKI on the development, survival, and enzymatic activity of Anagasta kuehniella larvae were evaluated, by incorporating AcKI in an artificial diet. Bioassays revealed a reduction in larval weight of ∼50% with the lower concentration of AcKI used in the study (0.5%). Although additionalassays showed an increase in endogenous trypsin and chymotrypsin activities, with a degree of AcKI-insensivity, AcKI produces an anti nutritional effect on A. kuehniella, indicating AcKI as a promising bioinsecticide protein for engineering plants that are resistant to insect pests.

Isolation, characterization and antifungal activity of proteinase inhibitors from Capsicum chinense Jacq. Seeds.

Capsicum species belong to the Solanaceae family and have great social, economic and agronomical significance. The present research presents data on the isolation and characterization of Capsicum chinense Jacq. peptides which were scrutinized in relation to their toxicity towards a diverse set of yeast species. The protein extract was separated with C18 reverse-phase chromatography in high performance liquid chromatography, resulting in three different peptide enriched fractions (PEFs) termed PEF1, PEF2 and PEF3. Tricine-SDS-PAGE of the PEF2 revealed peptides with molecular masses of approximately 5.0 and 8.5 kDa. These PEFs also exhibited strong antifungal activity against different yeasts. In the presence of the PEF2, Candida tropicalis exhibited morphological changes, including cellular agglomeration and formation of pseudohyphae. Determined N-terminal sequences of PEF2 and PEF3 were proven to be highly homologous to serine proteinase inhibitors, when analysed by comparative database sequence tools. For this reason were performed protease inhibitory activity assay. The PEFs displayed high inhibitory activity against trypsin and low inhibitory activity against chymotrypsin. PEF2 and PEF3 were considerably unsusceptible to a broad interval of pH and temperatures. Due to the myriad of application of Proteinase inhibitors (PIs) in fields ranging from plant protection against pathogens and pests to medicine such as in cancer and virus replication inhibition, the discovery of new PIs with new properties are of great interest.

Antimicrobial peptides from Adenanthera pavonina L. seeds: characterization and antifungal activity.

In this study, the antifungal activity of peptides extracted from Adenanthera pavonina seeds was assessed. Peptides were extracted and fractionated by DEAE-Sepharose chromatography. The non-retained D1 fraction efficiently inhibited the growth of the pathogenic fungi. This fraction was later further fractionated by reversed-phase chromatography, resulting in 23 sub-fractions. All separation processes were monitored by tricine-SDS-PAGE. Fractions H11 and H22 strongly inhibited the growth of Saccharomyces cerevisiae and Candida albicans. Fraction H11 caused 100% death in S. cerevisiae in an antimicrobial assay. The complete amino acid sequence of the peptide in fraction P2 was determined, revealing homology to plant defensins, which was named ApDef1. Peptides from fraction H22 were also sequenced.

Passion fruit flowers: Kunitz trypsin inhibitors and cystatin differentially accumulate in developing buds and floral tissues.

In order to better understand the physiological functions of protease inhibitors (PIs) the PI activity in buds and flower organs of passion fruit (Passiflora edulis Sims) was investigated. Trypsin and papain inhibitory activities were analyzed in soluble protein extracts from buds at different developmental stages and floral tissues in anthesis. These analyses identified high levels of inhibitory activity against both types of enzymes at all bud stages. Intriguingly, the inhibitory activity against both proteases differed remarkably in some floral tissues. While all organs tested were very effective against trypsin, only sepal and petal tissues exhibited strong inhibitory activity against papain. The sexual reproductive tissues (ovary, stigma-style and stamen) showed either significantly lower activity against papain or practically none. Gelatin-SDS-PAGE assay established that various trypsin inhibitors (TIs) homogenously accumulated in developing buds, although some were differentially present in floral organs. The N-terminal sequence analysis of purified inhibitors from stamen demonstrated they had homology to the Kunitz family of serine PIs. Western-blot analysis established presence of a ∼60 kDa cystatin, whose levels progressively increased during bud development. A positive correlation between this protein and strong papain inhibitory activity was observed in buds and floral tissues, except for the stigma-style. Differences in temporal and spatial accumulation of both types of PIs in passion fruit flowers are thus discussed in light of their potential roles in defense and development.

Identification of critical amino acids in the IgE epitopes of Ric c 1 and Ric c 3 and the application of glutamic acid as an IgE blocker.

BACKGROUND: The allergenicity of Ricinus communis L. (castor bean, Euphorbiaceae) is associated with components of its seeds and pollen. Castor bean allergy has been described not only in laboratory workers, but also in personnel working in oil processing mills, fertilizer retail, the upholstery industry and other industrial fields. In the present study, we describe the critical amino acids in the IgE-binding epitopes in Ric c 1 and Ric c 3, two major allergens of R. communis. In addition, we also investigate the cross-reactivity between castor bean and some air and food allergen extracts commonly used in allergy diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: The IgE reactivity of human sera from atopic patients was screened by immune-dot blot against castor bean allergens. Allergenic activity was evaluated in vitro using a rat mast cell activation assay and by ELISA. Cross-reactivity was observed between castor bean allergens and extracts from shrimp, fish, gluten, wheat, soybean, peanut, corn, house dust, tobacco and airborne fungal allergens. We observed that treatment of rat and human sera (from atopic patients) with glutamic acid reduced the IgE-epitope interaction. CONCLUSIONS/SIGNIFICANCE: The identification of glutamic acid residues with critical roles in IgE-binding to Ric c 3 and Ric c 1 support the potential use of free amino acids in allergy treatment.