Anticandidal activity of synthetic peptides: Mechanism of action revealed by scanning electron and fluorescence microscopies and synergism effect with nystatin.

Candida albicans has emerged as a major public health problem in recent decades. The most important contributing factor is the rapid increase in resistance to conventional drugs worldwide. Synthetic antimicrobial peptides (SAMPs) have attracted substantial attention as alternatives and/or adjuvants in therapeutic treatments due to their strong activity at low concentrations without apparent toxicity. Here, two SAMPs, named Mo-CBP3 -PepI (CPAIQRCC) and Mo-CBP3 -PepII (NIQPPCRCC), are described, bioinspired by Mo-CBP3 , which is an antifungal chitin-binding protein from Moringa oleifera seeds. Furthermore, the mechanism of anticandidal activity was evaluated as well as their synergistic effects with nystatin. Both peptides induced the production of reactive oxygen species (ROS), cell wall degradation, and large pores in the C. albicans cell membrane. In addition, the peptides exhibited high potential as adjuvants because of their synergistic effects, by increasing almost 50-fold the anticandidal activity of the conventional antifungal drug nystatin. These peptides have excellent potential as new drugs and/or adjuvants to conventional drugs for treatment of clinical infections caused by C. albicans.

Identification, characterization, and expression analysis of cowpea (Vigna unguiculata [L.] Walp.) miRNAs in response to cowpea severe mosaic virus (CPSMV) challenge.

KEY MESSAGE: Cowpea miRNAs and Argonaute genes showed differential expression patterns in response to CPSMV challenge Several biotic stresses affect cowpea production and yield. CPSMV stands out for causing severe negative impacts on cowpea. Plants have two main induced immune systems. In the basal system (PTI, PAMP-triggered immunity), plants recognize and respond to conserved molecular patterns associated with pathogens (PAMPs). The second type (ETI, Effector-triggered immunity) is induced after plant recognition of specific factors from pathogens. RNA silencing is another important defense mechanism in plants. Our research group has been using biochemical and proteomic approaches to learn which proteins and pathways are involved and could explain why some cowpea genotypes are resistant whereas others are susceptible to CPSMV. This current study was conducted to determine the role of cowpea miRNA in the interaction between a resistant cowpea genotype (BRS-Marataoã) and CPSMV. Previously identified and deposited plant microRNA sequences were used to find out all possible microRNAs in the cowpea genome. This search detected 617 mature microRNAs, which were distributed in 89 microRNA families. Next, 4 out of these 617 miRNAs and their possible target genes that encode the proteins Kat-p80, DEAD-Box, GST, and SPB9, all involved in the defense response of cowpea to CPSMV, had their expression compared between cowpea leaves uninoculated and inoculated with CPSMV. Additionally, the differential expression of genes that encode the Argonaute (AGO) proteins 1, 2, 4, 6, and 10 is reported. In summary, the studied miRNAs and AGO 2 and AGO4 associated genes showed differential expression patterns in response to CPSMV challenge, which indicate their role in cowpea defense.

Gene expression during development and overexpression after Cercospora kikuchii and salicylic acid challenging indicate defensive roles of the soybean toxin.

KEY MESSAGE: SBTX has defensive role against C. kikuchii, and therefore, its constituent genes SBTX17 and SBTX27 are promising candidates to engineer pathogen resistant plants. Soybean (Glycine max [L.] Merr.) is economically the most important legume crop in the world. Its productivity is strongly affected by fungal diseases, which reduce soybean production and seed quality and cause losses of billions of dollars worldwide. SBTX is a protein that apparently takes part in the defensive chemical arsenal of soybean against pathogens. This current study provides data that reinforce this hypothesis. Indeed, SBTX inhibited in vitro the mycelial growth of Cercospora kikuchii, it is constitutively located in the epidermal region of the soybean seed cotyledons, and it is exuded from mature imbibed seeds. Moreover, RT-qPCR analysis of the SBTX associated genes, SBTX17 and SBTX27, which encode for the 17 and 27 kDa polypeptide chains, showed that both genes are expressed in all studied plant tissues during the soybean development, with the highest levels found in the mature seeds and unifoliate leaves. In addition, to assess a local response of the soybean secondary leaves from 35-day-old plants, they were inoculated with C. kikuchii and treated with salicylic acid. It was verified using RT-qPCR that SBTX17 and SBTX27 genes overexpressed in leaves compared to controls. These findings strongly suggest that SBTX has defensive roles against C. kikuchii. Therefore, SBTX17 and SBTX27 genes are promising candidates to engineer pathogen resistant plants.

Antidermatophytic activity of synthetic peptides: Action mechanisms and clinical application as adjuvants to enhance the activity and decrease the toxicity of Griseofulvin.

BACKGROUND: Dermatophytes belonging to the Trichophyton genus are important human pathogens, but they have developed resistance to griseofulvin, the most common antifungal drug used to treat dermatophytosis. OBJECTIVE: This study was aimed to evaluate the antidermatophytic activity of synthetic peptides, as well as mechanisms of action and synergistic effect with griseofulvin. METHODS: Scanning electron microscopy (SEM), atomic force microscopy (AFM) and fluorescence microscopy (FM) were employed to understand the activity and the mechanism of action of peptides. RESULTS: Here we report that synthetic peptides at 50 µg/mL, a concentration 20-fold lower than griseofulvin, reduced the microconidia viability of T. mentagrophytes and T. rubrum by 100%, whereas griseofulvin decreased their viability by only 50% and 0%, respectively. The action mechanism of peptides involved cell wall damage, membrane pore formation and loss of cytoplasmic content. Peptides also induced overproduction of reactive oxygen species (ROS) and enhanced the activity of griseofulvin 10-fold against both fungi, suggesting synergistic effects, and eliminated the toxicity of this drug to human erythrocytes. Docking analysis revealed ionic and hydrophobic interactions between peptides and griseofulvin, which may explain the decline of griseofulvin toxicity when mixed with peptides. CONCLUSION: Therefore, our results strongly suggest six peptides with high potential to be employed alone as new drugs or as adjuvants to enhance the activity and decrease the toxicity of griseofulvin.

Gene expression and spatiotemporal localization of antifungal chitin-binding proteins during Moringa oleifera seed development and germination.

MAIN CONCLUSION: Chitin-binding proteins behave as storage and antifungal proteins in the seeds of Moringa oleifera. Moringa oleifera is a tropical multipurpose tree. Its seed constituents possess coagulant, bactericidal, fungicidal, and insecticidal properties. Some of these properties are attributed to a group of polypeptides denominated M. oleifera chitin-binding proteins (in short, Mo-CBPs). Within this group, Mo-CBP2, Mo-CBP3, and Mo-CBP4 were previously purified to homogeneity. They showed high amino acid similarity with the 2S albumin storage proteins. These proteins also presented antimicrobial activity against human pathogenic yeast and phytopathogenic fungi. In the present study, the localization and expression of genes that encode Mo-CBPs and the biosynthesis and degradation of the corresponding proteins during morphogenesis and maturation of M. oleifera seeds at 15, 30, 60, and 90 days after anthesis (DAA) and germination, respectively, were assessed. The Mo-CBP transcripts and corresponding proteins were not detected at 15 and 30 days after anthesis (DAA). However, they accumulated at the latter stages of seed maturation (60 and 90 DAA), reaching the maximum level at 60 DAA. The degradation kinetics of Mo-CBPs during seed germination by in situ immunolocalization revealed a reduction in the protein content 48 h after sowing (HAS). Moreover, Mo-CBPs isolated from seeds at 60 and 90 DAA prevented the spore germination of Fusarium spp. Taken together, these results suggest that Mo-CBPs play a dual role as storage and defense proteins in the seeds of M. oleifera.

A Bowman-Birk Inhibitor from the Seeds of Luetzelburgia auriculata Inhibits Staphylococcus aureus Growth by Promoting Severe Cell Membrane Damage.

Staphylococcus aureus is a multidrug-resistant bacterium responsible for several cases of hospital-acquired infections, which constitute a global public health problem. The introduction of new healthcare strategies and/or the discovery of molecules capable of inhibiting the growth or killing S. aureus would have a huge impact on the treatment of S. aureus-mediated diseases. Herein, a Bowman-Birk protease inhibitor ( LzaBBI), with strong in vitro antibacterial activity against S. aureus, was purified to homogeneity from Luetzelburgia auriculata seeds. LzaBBI in its native form is a 14.3 kDa protein and has a pI of 4.54, and its NH2-terminal sequence has high identity with other Bowman-Birk inhibitors. LzaBBI showed a mixed-type inhibitory activity against both trypsin and chymotrypsin, respectively, and it remained stable after both boiling at 98 °C for 120 min and incubation at various pHs. Scanning electron microscopy revealed that LzaBBI disrupted the S. aureus membrane integrity, leading to bacterial death. This study suggests that LzaBBI is a powerful candidate for developing a new antimicrobial to overcome drug resistance toward reducing hospital-acquired infections caused by S. aureus.

Photosynthetic and biochemical mechanisms of an EMS-mutagenized cowpea associated with its resistance to cowpea severe mosaic virus.

KEY MESSAGE: The seed treatment of a CPSMV-susceptible cowpea genotype with the mutagenic agent EMS generated mutagenized resistant plantlets that respond to the virus challenge by activating biochemical and physiological defense mechanisms. Cowpea is an important crop that makes major nutritional contributions particularly to the diet of the poor population worldwide. However, its production is low, because cowpea is naturally exposed to several abiotic and biotic stresses, including viral agents. Cowpea severe mosaic virus (CPSMV) drastically affects cowpea grain production. This study was conducted to compare photosynthetic and biochemical parameters of a CPSMV-susceptible cowpea (CE-31 genotype) and its derived ethyl methanesulfonate-mutagenized resistant plantlets, both challenged with CPSMV, to shed light on the mechanisms of virus resistance. CPSMV inoculation was done in the fully expanded secondary leaves, 15 days after planting. At 7 days post-inoculation, in vivo photosynthetic parameters were measured and leaves collected for biochemical analysis. CPSMV-inoculated mutagenized-resistant cowpea plantlets (MCPI) maintained higher photosynthesis index, chlorophyll, and carotenoid contents in relation to the susceptible (CE-31) CPSMV-inoculated cowpea (CPI). Visually, the MCPI leaves did not exhibit any viral symptoms neither the presence of the virus as examined by RT-PCR. In addition, MCPI showed higher SOD, GPOX, chitinase, and phenylalanine ammonia lyase activities, H2O2, phenolic contents, and cell wall lignifications, but lower CAT and APX activities in comparison to CPI. All together, these photosynthetic and biochemical changes might have contributed for the CPSMS resistance of MCPI. Contrarily, CPI plantlets showed CPSMV accumulation, severe disease symptoms, reduction in the photosynthesis-related parameters, chlorophyll, carotenoid, phenolic compound, and H2O2 contents, in addition to increased ß-1,3-glucanase, and catalase activities that might have favored viral infection.

A Protein Isolate from Moringa oleifera Leaves Has Hypoglycemic and Antioxidant Effects in Alloxan-Induced Diabetic Mice.

Moringa oleifera has been used in traditional medicine to treat diabetes. However, few studies have been conducted to relate its antidiabetic properties to proteins. In this study, a leaf protein isolate was obtained from M. oleifera leaves, named Mo-LPI, and the hypoglycemic and antioxidant effects on alloxan-induced diabetic mice were assessed. Mo-LPI was obtained by aqueous extraction, ammonium sulphate precipitation and dialysis. The electrophoresis profile and proteolytic hydrolysis confirmed its protein nature. Mo-LPI showed hemagglutinating activity, cross-reaction with anti-insulin antibodies and precipitation after zinc addition. Single-dose intraperitoneal (i.p.) administration of Mo-LPI (500 mg/kg·bw) reduced the blood glucose level (reductions of 34.3%, 60.9% and 66.4% after 1, 3 and 5 h, respectively). The effect of Mo-LPI was also evidenced in the repeated dose test with a 56.2% reduction in the blood glucose level on the 7th day after i.p. administration. Mo-LPI did not stimulate insulin secretion in diabetic mice. Mo-LPI was also effective in reducing the oxidative stress in diabetic mice by a decrease in malondialdehyde level and increase in catalase activity. Mo-LPI (2500 mg/kg·bw) did not cause acute toxicity to mice. Mo-LPI is a promising alternative or complementary agent to treat diabetes.

Label-free Proteomic Reveals that Cowpea Severe Mosaic Virus Transiently Suppresses the Host Leaf Protein Accumulation During the Compatible Interaction with Cowpea (Vigna unguiculata [L.] Walp.).

Viruses are important plant pathogens that threaten diverse crops worldwide. Diseases caused by Cowpea severe mosaic virus (CPSMV) have drawn attention because of the serious damages they cause to economically important crops including cowpea. This work was undertaken to quantify and identify the responsive proteins of a susceptible cowpea genotype infected with CPSMV, in comparison with mock-inoculated controls, using label-free quantitative proteomics and databanks, aiming at providing insights on the molecular basis of this compatible interaction. Cowpea leaves were mock- or CPSMV-inoculated and 2 and 6 days later proteins were extracted and analyzed. More than 3000 proteins were identified (data available via ProteomeXchange, identifier PXD005025) and 75 and 55 of them differentially accumulated in response to CPSMV, at 2 and 6 DAI, respectively. At 2 DAI, 76% of the proteins decreased in amount and 24% increased. However, at 6 DAI, 100% of the identified proteins increased. Thus, CPSMV transiently suppresses the synthesis of proteins involved particularly in the redox homeostasis, protein synthesis, defense, stress, RNA/DNA metabolism, signaling, and other functions, allowing viral invasion and spread in cowpea tissues.

Proteomic analysis and purification of an unusual germin-like protein with proteolytic activity in the latex of Thevetia peruviana.

MAIN CONCLUSION: The latex from Thevetia peruviana is rich in plant defense proteins, including a 120 kDa cysteine peptidase with structural characteristics similar to germin-like proteins. More than 20,000 plant species produce latex, including Apocynaceae, Sapotaceae, Papaveraceae and Euphorbiaceae. To better understand the physiological role played by latex fluids, a proteomic analysis of Thevetia peruviana (Pers.) Schum latex was performed using two-dimensional gel electrophoresis and mass spectrometry. A total of 33 proteins (86 %) were identified, including storage proteins, a peptidase inhibitor, cysteine peptidases, peroxidases and osmotins. An unusual cysteine peptidase, termed peruvianin-I, was purified from the latex by a single chromatographic step involving gel filtration. The enzyme (glycoprotein) was inhibited by E-64 and iodoacetamide and exhibited high specific activity towards azocasein (K m 17.6 µM), with an optimal pH and temperature of 5.0-6.0 and 25-37 °C, respectively. Gel filtration chromatography, two-dimensional gel electrophoresis, and mass spectrometry revealed that peruvianin-I possesses 120 kDa, pI 4.0, and six subunits (20 kDa). A unique N-terminal amino acid sequence was obtained to oligomer and monomers of peruvianin-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36). High-resolution images from atomic force microscopy showed the homohexameric structure of peruvianin-I may be organized as a trimer of dimers that form a central channel similar to germin-like proteins. Peruvianin-I exhibited no oxalate oxidase and superoxide dismutase activity or antifungal effects. Peruvianin-I represents the first germin-like protein (GLP) with cysteine peptidase activity, an activity unknown in the GLP family so far.

A 2S Albumin from the Seed Cake of Ricinus communis Inhibits Trypsin and Has Strong Antibacterial Activity against Human Pathogenic Bacteria.

Hospital-acquired infections caused by antibiotic-resistant bacteria threaten the lives of many citizens all over the world. Discovery of new agents to hinder bacterial development would have a significant impact on the treatment of infections. Here, the purification and characterization of Rc-2S-Alb, a protein that belongs to the 2S albumin family, from Ricinus communis seed cake, are reported. Rc-2S-Alb was purified after protein extraction with Tris-HCl buffer, pH 7.5, fractionation by ammonium sulfate (50-75%), and chromatography on Phenyl-Sepharose and DEAE-Sepharose. Rc-2S-Alb, a 75 kDa peptide, displays trypsin inhibitory activity and has high in vitro antibacterial activity against Bacillus subtilis, Klebsiella pneumonia, and Pseudomonas aeruginosa, which are important human pathogenic bacteria. Atomic force microscopy studies indicated that Rc-2S-Alb disrupts the bacterial membrane with loss of the cytoplasm content and ultimately bacterial death. Therefore, Rc-2S-Alb is a powerful candidate for the development of an alternative drug that may help reduce hospital-acquired infections.

An antifungal peptide from Coffea canephora seeds with sequence homology to glycine-rich proteins exerts membrane permeabilization and nuclear localization in fungi.

BACKGROUND: The superfamily of glycine-rich proteins (GRPs) corresponds to a large and complex group of plant proteins that may be involved in many developmental and physiological processes such as RNA biogenesis, stress tolerance, pollen hydration and plant-pathogen interactions, showing defensive activity against fungi, bacteria and viruses. METHODS: In this study, the peptides from Coffea canephora seeds were extracted according to the methods of Egorov et al. (2005). The purified peptide was submitted for amino acid sequencing and antimicrobial activity measurement. RESULTS: The purified peptide with a molecular weight of 7kDa, named Cc-GRP, was observed to display homology to GRPs. The Cc-GRP-fungi interaction led to morphological changes and membrane permeability, including the formation of pseudohyphae, which were visualized with the aid of SYTOX green dye. Additionally, Cc-GRP also prevented colony formation by yeasts. Antifungal assays of Fusarium oxysporum and Colletotrichum lindemuthianum, observed by light microscopy, showed that the two molds exhibited morphological changes after the growth assay. Cc-GRP coupled to FITC and its subsequent treatment with DAPI revealed the presence of the peptide in the cell wall, cell surface and nucleus of F. oxysporum. CONCLUSIONS AND GENERAL SIGNIFICANCE: In this work we purified, characterized and evaluated the in vitro effect on fungi of a new peptide from coffee, named Cc-GRP, which is involved in the plant defense system against pathogens by acting through a membrane permeabilization mechanism and localized in the nuclei of fungal cells. We also showed, for the first time, the intracellular localization of Cc-GRP during antimicrobial assay.

Vicilin-like peptides from Capsicum baccatum L. seeds are α-amylase inhibitors and exhibit antifungal activity against important yeasts in medical mycology.

The objective of this study was to isolate antimicrobial peptides from Capsicum baccatum seeds and evaluate their antimicrobial activity and inhibitory effects against α-amylase. Initially, proteins from the flour of C. baccatum seeds were extracted in sodium phosphate buffer, pH 5.4, and precipitated with ammonium sulfate at 90% saturation. The D1 and D2 fractions were subjected to antifungal tests against the yeasts Saccharomyces cerevisiae, Candida albicans, Candida tropicalis, and Kluyveromyces marxiannus, and tested against α-amylases from Callosobruchus maculates and human saliva. The D2 fraction presented higher antimicrobial activity and was subjected to further purification and seven new different fractions (H1-H7) were obtained. Peptides in the H4 fraction were sequenced and the N-terminal sequences revealed homology with previously reported storage vicilins from seeds. The H4 fraction exhibited strong antifungal activity and also promoted morphological changes in yeast, including pseudohyphae formation. All fractions, including H4, inhibited mammalian α-amylase activity but only the H4 fraction was able to inhibit C. maculatus α-amylase activity. These results suggest that the fractions isolated from the seeds of C. baccatum can act directly in plant defenses against pathogens and insects.

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.

Selection of suitable soybean EF1α genes as internal controls for real-time PCR analyses of tissues during plant development and under stress conditions.

KEY MESSAGE: The EF1α genes were stable in the large majority of soybean tissues during development and in specific tissues/conditions under stress. Quantitative real-time PCR (qPCR) analysis strongly depends on transcript normalization using stable reference genes. Reference genes are generally encoded by multigene families and are used in qPCR normalization; however, little effort has been made to verify the stability of different gene members within a family. Here, the expression stability of members of the soybean EF1α gene family (named EF1α 1a1, 1a2, 1b, 2a, 2b and 3) was evaluated in different tissues during plant development and stress exposure (SA and PEG). Four genes (UKN1, SKIP 16, EF1ß and MTP) already established as stably expressed were also used in the comparative analysis. GeNorm analyses revealed different combinations of reference genes as stable in soybean tissues during development. The EF1α genes were the most stable in cotyledons (EF1α 3 and EF1α 1b), epicotyls (EF1α 1a2, EF1α 2b and EF1α 1a1), hypocotyls (EF1α 1a1 and EF1ß), pods (EF1α 2a and EF1α 2b) and roots (EF1α 2a and UKN1) and less stable in tissues such as trifoliate and unifoliate leaves and germinating seeds. Under stress conditions, no suitable combination including only EF1α genes was found; however, some genes were relatively stable in leaves (EF1α 1a2) and roots (EF1α 1a1) treated with SA as well as in roots treated with PEG (EF1α 2b). EF1α 2a was the most stably expressed EF1α gene in all soybean tissues under stress. Taken together, our data provide guidelines for the selection of EF1α genes for use as reference genes in qPCR expression analyses during plant development and under stress conditions.

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves.

BACKGROUND: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). METHODS: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. RESULTS: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56­4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% ß-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx. GENERAL SIGNIFICANCE: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.

New small proteinase inhibitors from Capsicum annuum seeds: Characterization, stability, spectroscopic analysis and a cDNA cloning.

Recent results from our laboratory have previously shown the purification of a small serine proteinase inhibitor (PI), named CaTI1, from Capsicum annuum seeds. This work demonstrated the characterization of CaTI now named CaTI1, and the identification of two other small serine PIs, named CaTI2 and CaTI3, also present in these seeds. CaTI1 presented molecular mass of 6 kDa and pI value of ∼9.0. CaTI1 inhibited both trypsin and chymotrypsin with inhibition constants (Ki and Ki') of 14 and 2.8 nM for trypsin and 4.3 and 0.58 nM for chymotrypsin, respectively. Circular dichroism analysis suggested the predominance of both disordered and ß-strands regions in the secondary structure. CaTI1 presented striking physico-chemical stability. In an attempt to get the entire sequence of CaTI1 we found another PI called CaTI2. The discussion of this finding is in the main text. A degenerate primer was designed based on the sequence of trypsin inhibitor CaTI1 in an attempt to achieve the cloning of this PI. Surprisingly, the alignment of the predicted peptide derived from the cDNA with the protein database showed similarity with other C. annuun PIs, and thus it was called CaTI3.

Screening of antimicrobials from Caribbean sea animals and isolation of bactericidal proteins from the littoral mollusk Cenchritis muricatus.

Marine organisms represent approximately half of the world's biodiversity by virtue of the sea being an immense reservoir of bioactive molecules. Here, antimicrobial crude extract activities of different marine invertebrates from the Caribbean Sea were evaluated. One of the most active, crude extracts was that marine snail Cenchritis muricatus, it was capable of totally inhibiting the development of Staphylococcus aureus and also showed a growth inhibition of 95.9% in Escherichia coli. Aiming to isolate molecules that confirm antimicrobial activity, the crude extract was purified by reversed-phase HPLC C-18 chromatography. Thereafter, one of the obtained fractions preserved this antibacterial activity. Furthermore, SDS-PAGE analysis (15%) showed the presence of two proteins of molecular masses with approximately 10 and 15 kDa, respectively. The first 19 amino acids of both proteins were sequenced by using Edman degradation, yielding unidentified primary structures compared against sequences deposited at NCBI databank. This is the first report of antibacterial proteins isolated from the mollusk Cenchritis muricatus and these proteins could be used as antibiotic alternatives in the aquacultural industry, as well as in agricultural or biomedical research.

A brief report on some health aspects of rats fed with crescent levels of recombinant chagasin, a potential plant defense protein.

Chagasin may be considered a potential plant-incorporated protectant (PIP) protein due to its deleterious effects on insect pests. However, extensive safety studies with PIP's are necessary before introducing them into the target plant. Thus, a short-term feeding trial in rats with high doses of r-chagasin was conducted to provide evidences about its safety. Three test diets containing casein + r-chagasin (0.25, 0.5 and 1% of total protein) were offered to rats (10 days). The test diets did not show adverse effects upon the development, organ weight, hematological parameters and serum protein profiles of rats, providing preliminary information on the safety of r-chagasin.

Chitinase from the Latex of Ficus benjamina L. Displays Antifungal Activity by Inducing ROS Generation and Structural Damage to the Fungal Cell Wall and Plasma Membrane.

BACKGROUND: Chitinases are plant defense-related proteins with a high biotechnological potential to be applied in agriculture. OBJECTIVES: This study aimed to purify a chitinase from the latex of Ficus benjamina. METHODS: An antifungal class I chitinase, named FbLx-Chi-1, was purified from the latex of Ficus benjamina after precipitation with 30-60% ammonium sulfate and affinity chromatography on a chitin column and antifungal potential assay against phytopathogenic fungi important to agriculture. RESULTS: FbLx-Chi-1 has 30 kDa molecular mass, as estimated by SDS-PAGE and the optimal pH and temperature for full chitinolytic activity were 5.5 and 60ºC, respectively. FbLx-Chi-1 is a high pH-, ion-tolerant and thermostable protein. Importantly, FbLx-Chi-1 hindered the growth of the phytopathogenic fungi Colletotrichum gloeosporioides, Fusarium pallidoroseum, and Fusarium oxysporum. The action mode of FbLx-Chi-1 to hamper F. pallidoroseum growth seems to be correlated with alterations in the morphology of the hyphal cell wall, increased plasma membrane permeability, and overproduction of reactive oxygen species. CONCLUSION: These findings highlight the biotechnological potential of FbLx-Chi-1 to control important phytopathogenic fungi in agriculture. In addition, FbLx-Chi-1 could be further explored to be used in industrial processes such as the large-scale environmentally friendly enzymatic hydrolysis of chitin to produce its monomer N-acetyl-ß-D-glucosamine, which is employed for bioethanol production, in cosmetics, in medicine, and for other multiple applications.