Publications by authors named "Carlos Bloch"

74 Publications

Identification of Differential N-Glycan Compositions in the Serum and Tissue of Colon Cancer Patients by Mass Spectrometry.

Biology (Basel) 2021 Apr 20;10(4). Epub 2021 Apr 20.

Division of Colorectal Surgery, University Hospital of Brasilia, School of Medicine, University of Brasilia, SGAN 605, Brasilia-DF 70840-901, Brazil.

Colorectal cancer (CRC) ranks second as the leading cause of cancer-related deaths worldwide. N-glycosylation is one of the most common posttranslational protein modifications. Therefore, we studied the total serum N-glycome (TSNG) of 13 colon cancer patients compared to healthy controls using MALDI-TOF/MS and LC-MS. N-glycosylation of cancer tumor samples from the same cohort were further quantified using a similar methodology. In total, 23 N-glycan compositions were down-regulated in the serum of colon cancer patients, mostly galactosylated forms whilst the mannose-rich HexNAc2Hex7, the fucosylated bi-antennary glycan HexNAc4Hex5Fuc1NeuAc2, and the tetra-antennary HexNAc6Hex7NeuAc3 were up-regulated in serum. Hierarchical clustering analysis of TSNG correctly singled out 85% of the patients from controls. Albeit heterogenous, N-glycosylation of tumor samples showed overrepresented oligomannosidic, bi-antennary hypogalactosylated, and branched compositions related to normal colonic tissue, in both MALDI-TOF/MS and LC-MS analysis. Moreover, compositions found upregulated in tumor tissue were mostly uncorrelated to compositions in serum of cancer patients. Mass spectrometry-based N-glycan profiling in serum shows potential in the discrimination of patients from healthy controls. However, the compositions profile in serum showed no parallel with N-glycans in tumor microenvironment, which suggests a different origin of compositions found in serum of cancer patients.
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http://dx.doi.org/10.3390/biology10040343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8074232PMC
April 2021

Head-to-Tail Cyclization after Interaction with Trypsin: A Scorpion Venom Peptide that Resembles Plant Cyclotides.

J Med Chem 2020 09 12;63(17):9500-9511. Epub 2020 Aug 12.

Neuropharma Lab, Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Brası́lia, Brasília-DF 70910-900, Brazil.

Peptidase inhibitors (PIs) have been broadly studied due to their wide therapeutic potential for human diseases. A potent trypsin inhibitor from scorpion venom was characterized and named ToPI1, with 33 amino acid residues and three disulfide bonds. The X-ray structure of the ToPI1:trypsin complex, in association with the mass spectrometry data, indicate a sequential set of events: the complex formation with the inhibitor Lys in the trypsin S1 pocket, the inhibitor C-terminal residue Ser cleavage, and the cyclization of ToPI1 via a peptide bond between residues Ile and Lys. Kinetic and thermodynamic characterization of the complex was obtained. ToPI1 shares no sequence similarity with other PIs characterized to date and is the first PI with CS-α/β motif described from animal venoms. In its cyclic form, it shares structural similarities with plant cyclotides that also inhibit trypsin. These results bring new insights for studies with venom compounds, PIs, and drug design.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00686DOI Listing
September 2020

Phylloseptin-1 is Leishmanicidal for Amastigotes of Inside Infected Macrophages.

Int J Environ Res Public Health 2020 07 6;17(13). Epub 2020 Jul 6.

Núcleo de Pesquisa em Morfologia e Imunologia Aplicada, NuPMIA, Área de Morfologia, Faculdade de Medicina, FM, Universidade de Brasília, Brasília, DF 70910900, Brazil.

Leishmania protozoans are the causal agents of neglected diseases that represent an important public health issue worldwide. The growing occurrence of drug-resistant strains of and severe side effects of available treatments represent an important challenge for the leishmaniases treatment. We have previously reported the leishmanicidal activity of phylloseptin-1 (PSN-1), a peptide found in the skin secretion of (=), against promastigotes. However, its impact on the amastigote form of and its impact on infected macrophages are unknown. In this work, we evaluated the effects of PSN-1 on amastigotes of inside macrophages infected in vitro. We assessed the production of hydrogen peroxide and nitric oxide, as well as the levels of inflammatory and immunomodulatory markers (TGF-β, TNF-α and IL-12), in infected and non-infected macrophages treated with PSN-1. Treatment with PSN-1 decreased the number of infected cells and the number of ingested amastigotes per cell when compared with the untreated cells. At 32 µM (64 µg/mL), PSN-1 reduced hydrogen peroxide levels in both infected and uninfected macrophages, whereas it had little effect on NO production or TGF-β release. The effect of PSN-1 on IL-12 and TNF-α secretion depended on its concentration, but, in general, their levels tended to increase as PSN-1 concentration increased. Further in vitro and in vivo studies are needed to clarify the mechanisms of action of PSN-1 and its interaction with the immune system aiming to develop pharmacological applications.
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http://dx.doi.org/10.3390/ijerph17134856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370015PMC
July 2020

Intragenic antimicrobial peptides (IAPs) from human proteins with potent antimicrobial and anti-inflammatory activity.

PLoS One 2019 6;14(8):e0220656. Epub 2019 Aug 6.

Laboratório de Espectrometria de Massa, LEM, Embrapa Recursos Genéticos e Biotecnologia, Brasília, DF, Brasil.

Following the treads of our previous works on the unveiling of bioactive peptides encrypted in plant proteins from diverse species, the present manuscript reports the occurrence of four proof-of-concept intragenic antimicrobial peptides in human proteins, named Hs IAPs. These IAPs were prospected using the software Kamal, synthesized by solid phase chemistry, and had their interactions with model phospholipid vesicles investigated by differential scanning calorimetry and circular dichroism. Their antimicrobial activity against bacteria, yeasts and filamentous fungi was determined, along with their cytotoxicity towards erythrocytes. Our data demonstrates that Hs IAPs are capable to bind model membranes while attaining α-helical structure, and to inhibit the growth of microorganisms at concentrations as low as 1μM. Hs02, a novel sixteen residue long internal peptide (KWAVRIIRKFIKGFIS-NH2) derived from the unconventional myosin 1h protein, was further investigated in its capacity to inhibit lipopolysaccharide-induced release of TNF-α in murine macrophages. Hs02 presented potent anti-inflammatory activity, inhibiting the release of TNF-α in LPS-primed cells at the lowest assayed concentration, 0.1 μM. A three-dimensional solution structure of Hs02 bound to DPC micelles was determined by Nuclear Magnetic Resonance. Our work exemplifies how the human genome can be mined for molecules with biotechnological potential in human health and demonstrates that IAPs are actual alternatives to antimicrobial peptides as pharmaceutical agents or in their many other putative applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0220656PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684085PMC
March 2020

Whey hydrolysate-based ingredient with dual functionality: From production to consumer's evaluation.

Food Res Int 2019 08 27;122:123-128. Epub 2019 Mar 27.

Embrapa Agroindústria de Alimentos, Avenida das Américas, 29501, Rio de Janeiro, RJ 23020-470, Brazil.

The aim of the present study concerns the development, characterization and sensory evaluation of a dual-functional whey hydrolysate. Four concentrations of commercial pepsin (0.48%, 0.95%, 1.43%, 1.91% w/w) were evaluated. The hydrolyses curves and the Reversed-Phase High Performance Liquid Chromatography analyses showed a direct relationship between enzyme concentration and degree of hydrolysis. Through mass spectrometry 21 peptides were identified and 5 of them have never been described in the literature before. The hydrolysate produced (PC3) induced a vascular relaxation of 65.02% in phenylephrine-contracted rat aortic rings. PC3 powder presented a homogeneous aspect with a mean particle size of 86.39 μm, high water solubility (>92%) in a wide pH range (1-12) and an increase of 33% in oil absorption capacity, when compared to the unhydrolyzed product. Sensory analysis showed a high acceptance (7.6 in a 9-point hedonic scale) of the hydrolysate among 100 consumers. The results brought the possibility of developing a whey hydrolysate with high vasorelaxant activity, great technological properties and sensory appeal, as an interesting dual-functional ingredient to be incorporated into food products.
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http://dx.doi.org/10.1016/j.foodres.2019.03.060DOI Listing
August 2019

Identification and characterization of phospholipases A from the skin secretion of Pithecopus azureus anuran.

Toxicon 2019 Sep 4;167:10-19. Epub 2019 Jun 4.

Laboratório de Espectrometria de Massa, Embrapa Recursos Genéticos e Biotecnologia, Brazil. Electronic address:

The present work reports the isolation, characterization and the complete sequence of a phospholipase A (PLA) present in the skin secretion of Pithecopus azureus. Among several peptides and small proteins previously described by our group from some species belonging to this amphibian genus (formerly named Phyllomedusa), a 15 kDa N-glycosylated protein showing PLA activity was purified, assayed, sequenced and named Pa-PLA. The Pithecopus azureus skin phospholipase A polypeptide chain is composed by 125 amino acid residues linked by seven disulfide bonds and two N-glycosylated sites (N67 and N108). The Pa-PLA enzymatic activity was qualitatively evaluated and compared to classical viperid PLA showing that both, native and deglycosylated Pa-PLA forms, are catalytically functional. The tridimensional molecular model of Pa-PLA indicates that the observed glycan moieties are suggestively placed far from the active site of that enzyme and therefore having little or no significant role on the direct interaction of the Pa-PLA catalytic pocket and its substrates.
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http://dx.doi.org/10.1016/j.toxicon.2019.06.002DOI Listing
September 2019

A previously undescribed hexapeptide His-Arg-Phe-Leu-Arg-His-NH from amphibian skin secretion shows CO and metal biding affinities.

Peptides 2018 08 19;106:37-44. Epub 2018 Jun 19.

Laboratório de Espectrometria de Massa, Embrapa Recursos Genéticos e Biotecnologia, Brasília, Distrito Federal, Brazil. Electronic address:

A previously undescribed six residues long peptide His-Arg-Phe-Leu-Arg-His was identified and purified from the skin secretion of the amphibian Phyllomedusa centralis. A synthetic analogue carboxyamidated HRFLRH-NH showed structural changes induced by CO and metal ions in aqueous solution when analyzed by NMR. The present work reports NMR structures for the carboxyamidated hexapeptide in the presence CO, Zn and Cd, suggesting possible affinity regions on the polypeptide chain for each ligand. The NMR structures were optimized by DFT to identify probable biding sites of these species in the polypeptide structure. To our best knowledge, this is the first time that a putative CO binding site is described on a peptide structure obtained in aqueous conditions, at room temperature.
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http://dx.doi.org/10.1016/j.peptides.2018.06.003DOI Listing
August 2018

Imaging Mass Spectrometry of Endogenous Polypeptides and Secondary Metabolites from Galls Induced by Root-Knot Nematodes in Tomato Roots.

Mol Plant Microbe Interact 2018 10 29;31(10):1048-1059. Epub 2018 Aug 29.

4 INRA, Université Côte d'Azur, CNRS, ISA, 06903, Sophia Antipolis, France.

Nematodes are devastating pests that infect most cultivated plant species and cause considerable agricultural losses worldwide. The understanding of metabolic adjustments induced during plant-nematode interaction is crucial to generate resistant plants or to select more efficient molecules to fight against this pest. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been used herein for in situ detection and mapping endogenous polypeptides and secondary metabolites from nematode-induced gall tissue. One of the major critical features of this technique is sample preparation; mainly, the generation of intact sections of plant cells with their rigid cell walls and vacuolated cytoplasm. Our experimental settings allowed us to obtain sections without contamination of exogenous ions or diffusion of molecules and to map the differential presence of low and high molecular weight ions in uninfected roots compared with nematode-induced galls. We predict the presence of lipids in both uninfected roots and galls, which was validated by MALDI time-of-flight tandem mass spectrometry and high-resolution mass spectrometry analysis of lipid extracts. Based on the isotopic ion distribution profile, both esters and glycerophospholipids were predicted compounds and may be playing an important role in gall development. Our results indicate that the MALDI-MSI technology is a promising tool to identify secondary metabolites as well as peptides and proteins in complex plant tissues like galls to decipher molecular processes responsible for infection and maintenance of these feeding sites during nematode parasitism.
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http://dx.doi.org/10.1094/MPMI-02-18-0049-RDOI Listing
October 2018

The Antifungal Plant Defensin HsAFP1 Is a Phosphatidic Acid-Interacting Peptide Inducing Membrane Permeabilization.

Front Microbiol 2017 21;8:2295. Epub 2017 Nov 21.

Centre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium.

HsAFP1, a plant defensin isolated from coral bells (), is characterized by broad-spectrum antifungal activity. Previous studies indicated that HsAFP1 binds to specific fungal membrane components, which had hitherto not been identified, and induces mitochondrial dysfunction and cell membrane permeabilization. In this study, we show that HsAFP1 reversibly interacts with the membrane phospholipid phosphatidic acid (PA), which is a precursor for the biosynthesis of other phospholipids, and to a lesser extent with various phosphatidyl inositol phosphates (PtdInsP's). Moreover, via reverse ELISA assays we identified two basic amino acids in HsAFP1, namely histidine at position 32 and arginine at position 52, as well as the phosphate group in PA as important features enabling this interaction. Using a HsAFP1 variant, lacking both amino acids (HsAFP1[H32A][R52A]), we showed that, as compared to the native peptide, the ability of this variant to bind to PA and PtdInsP's is reduced (≥74%) and the antifungal activity of the variant is reduced (≥2-fold), highlighting the link between PA/PtdInsP binding and antifungal activity. Using fluorescently labelled HsAFP1 in confocal microscopy and flow cytometry assays, we showed that HsAFP1 accumulates at the cell surface of yeast cells with intact membranes, most notably at the buds and septa. The resulting HsAFP1-induced membrane permeabilization is likely to occur after HsAFP1's internalization. These data provide novel mechanistic insights in the mode of action of the HsAFP1 plant defensin.
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http://dx.doi.org/10.3389/fmicb.2017.02295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702387PMC
November 2017

A Linear 19-Mer Plant Defensin-Derived Peptide Acts Synergistically with Caspofungin against Biofilms.

Front Microbiol 2017 20;8:2051. Epub 2017 Oct 20.

Centre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium.

Public health problems are associated with device-associated biofilm infections, with being the major fungal pathogen. We previously identified potent antibiofilm combination treatment in which the antifungal plant defensin HsAFP1 is co-administered with caspofungin, the preferred antimycotic to treat such infections. In this study, we identified the smallest linear HsAFP1-derived peptide that acts synergistically with caspofungin or anidulafungin against as HsLin06_18, a 19-mer peptide derived from the C-terminal part of HsAFP1. The [caspofungin + HsLin06_18] combination significantly reduced biofilm formation of and on catheters, as well as biofilm formation of a caspofungin-resistant strain. The [caspofungin + HsLin06_18] combination was not cytotoxic and reduced biofilm formation of using a subcutaneous rat catheter model, as compared to control treatment. Mode of action research on the [caspofungin + HsLin06_18] combination pointed to caspofungin-facilitated HsLin06_18 internalization and immediate membrane permeabilization. All these findings point to broad-spectrum antibiofilm activity of a combination of HsLin06_18 and caspofungin.
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http://dx.doi.org/10.3389/fmicb.2017.02051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655031PMC
October 2017

Ruthenium(II) complexes of 1,3-thiazolidine-2-thione: Cytotoxicity against tumor cells and anti-Trypanosoma cruzi activity enhanced upon combination with benznidazole.

J Inorg Biochem 2016 Mar 2;156:153-63. Epub 2016 Jan 2.

Departamento de Química, Universidade Federal de São Carlos, CP 676, CEP 13565-905 São Carlos, SP, Brazil. Electronic address:

Three new mixed and mononuclear Ru(II) complexes containing 1,3-thiazolidine-2-thione (tzdtH) were synthesized and characterized by spectroscopic analysis, molar conductivity, cyclic voltammetry, high-resolution electrospray ionization mass spectra and X-ray diffraction. The complexes presented unique stereochemistry and the proposed formulae are: [Ru(tzdt)(bipy)(dppb)]PF6 (1), cis-[Ru(tzdt)2(PPh3)2] (2) and trans-[Ru(tzdt)(PPh3)2(bipy)]PF6 (3), where dppb=1,4-bis(diphenylphosphino)butane and bipy=2,2'-bipyridine. These complexes demonstrated strong cytotoxicity against cancer cell lines when compared to cisplatin. Specifically, complex 2 was the most potent cytotoxic agent against MCF-7 breast cells, while complexes 1 and 3 were more active in DU-145 prostate cells. Binding of complexes to ctDNA was determined by UV-vis titration and viscosity measurements and revealed binding constant (Kb) values in range of 1.0-4.9×10(3)M(-1), which are characteristic of compounds possessing weak affinity to ctDNA. In addition, these complexes presented antiparasitic activity against Trypanosoma cruzi. Specifically, complex 3 demonstrated strong potency, moderate selectivity index and acted in synergism with the approved antiparasitic drug, benznidazole. Additionally, complex 3 caused parasite cell death through a necrotic process. In conclusion, we demonstrated that Ru(II) complexes have powerful pharmacological activity, while the metal-free tzdtH does not provoke the same outcome.
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http://dx.doi.org/10.1016/j.jinorgbio.2015.12.024DOI Listing
March 2016

Secretome analysis of the mycoparasitic fungus Trichoderma harzianum ALL 42 cultivated in different media supplemented with Fusarium solani cell wall or glucose.

Proteomics 2016 Feb;16(3):477-90

Laboratório de Enzimologia, Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Goiás (ICB), Goiânia, GO, Brazil.

Trichoderma harzianum is a fungus well known for its potential as a biocontrol agent against many fungal phytopathogens. The aim of this study was to characterize the proteins secreted by T. harzianum ALL42 when its spores were inoculated and incubated for 48 h in culture media supplemented with glucose (GLU) or with cell walls from Fusarium solani (FSCW), a phytopathogen that causes severe losses in common bean and soy crops in Brazil, as well as other crop diseases around the world. Trichoderma harzianum was able to grow in Trichoderma Liquid Enzyme Production medium (TLE) and Minimal medium (MM) supplemented with FSCW and in TLE+GLU, but was unable to grow in MM+GLU medium. Protein quantification showed that TLE+FSCW and MM+FSCW had 45- and 30- fold, respectively, higher protein concentration on supernatant when compared to TLE+GLU, and this difference was observable on 2D gel electrophoresis (2DE). A total of 94 out of 105 proteins excised from 2DE maps were identified. The only protein observed in all three conditions was epl1. In the media supplemented with FSCW, different hydrolases such as chitinases, β-1,3-glucanases, glucoamylases, α-1,3-glucanases and proteases were identified, along with other proteins with no known functions in mycoparasitism, such as npp1 and cys. Trichoderma harzianum showed a complex and diverse arsenal of proteins that are secreted in response to the presence of FSCW, with novel proteins not previously described in mycoparasitic-related studies.
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http://dx.doi.org/10.1002/pmic.201400546DOI Listing
February 2016

Skin secretion peptides: the molecular facet of the deimatic behavior of the four-eyed frog, Physalaemus nattereri (Anura, Leptodactylidae).

Rapid Commun Mass Spectrom 2015 Nov;29(21):2061-8

Embrapa Recursos Genéticos e Biotecnologia, Brasília-DF, Brazil.

Rationale: Amphibians can produce a large amount of bioactive peptides over the skin. In order to map the precise tissue localization of these compounds and evaluate their functions, mass spectrometry imaging (MSI) and gene expression studies were used to investigate a possible correlation between molecules involved in the antimicrobial defense mechanisms and anti-predatory behavior by Physalaemus nattereri.

Methods: Total skin secretion of P. nattereri was analyzed by classical Protein Chemistry and proteomic techniques. Intact inguinal macroglands were dissected from the rest of the skin and both tissues were analyzed by MSI and real-time polymerase chain reaction (RT-PCR) experiments. Peptides were primarily identified by de novo sequencing, automatic Edman degradation and cDNA data.

Results: Fifteen bradykinin (BK)-related peptides and two antimicrobial peptides were sequenced and mapped by MSI on the inguinal macrogland and the rest of P. nattereri skin. RT-PCR results revealed that BK-related peptide levels of expression were about 30,000 times higher on the inguinal macroglands than on the any other region of the skin, whilst antimicrobial peptide ions appear to be evenly distributed in both investigated regions.

Conclusions: The presence of antimicrobial peptides in all investigated tissue regions is in accordance with the defensive role against microorganisms thoroughly demonstrated in the literature, whereas BK-related molecules are largely found on the inguinal macroglands suggesting an intriguing link between their noxious activities against potential predators of P. nattereri and the frog's deimatic behavior.
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http://dx.doi.org/10.1002/rcm.7313DOI Listing
November 2015

Proteomic analysis of free-living Bradyrhizobium diazoefficiens: highlighting potential determinants of a successful symbiosis.

BMC Genomics 2014 Aug 3;15:643. Epub 2014 Aug 3.

Embrapa Soja, Embrapa Soja, C,P, 231, 86001-970 Londrina, Paraná, Brazil.

Background: Strain CPAC 7 (=SEMIA 5080) was recently reclassified into the new species Bradyrhizobium diazoefficiens; due to its outstanding efficiency in fixing nitrogen, it has been used in commercial inoculants for application to crops of soybean [Glycine max (L.) Merr.] in Brazil and other South American countries. Although the efficiency of B. diazoefficiens inoculant strains is well recognized, few data on their protein expression are available.

Results: We provided a two-dimensional proteomic reference map of CPAC 7 obtained under free-living conditions, with the successful identification of 115 spots, representing 95 different proteins. The results highlighted the expression of molecular determinants potentially related to symbiosis establishment (e.g. inositol monophosphatase, IMPase), fixation of atmospheric nitrogen (N2) (e.g. NifH) and defenses against stresses (e.g. chaperones). By using bioinformatic tools, it was possible to attribute probable functions to ten hypothetical proteins. For another ten proteins classified as "NO related COG" group, we analyzed by RT-qPCR the relative expression of their coding-genes in response to the nodulation-gene inducer genistein. Six of these genes were up-regulated, including blr0227, which may be related to polyhydroxybutyrate (PHB) biosynthesis and competitiveness for nodulation.

Conclusions: The proteomic map contributed to the identification of several proteins of B. diazoefficiens under free-living conditions and our approach-combining bioinformatics and gene-expression assays-resulted in new information about unknown genes that might play important roles in the establishment of the symbiosis with soybean.
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http://dx.doi.org/10.1186/1471-2164-15-643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287336PMC
August 2014

MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex.

Mycologia 2014 Jul-Aug;106(4):865-78. Epub 2014 Jul 1.

USDA-ARS Biological Integrated Pest Management Research, Robert W. Holley Center for Agriculture and Health, 538 Tower Road, Ithaca, New York 14853

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenetic classification of species in the Metarhizium anisopliae complex. Initially the phylogenetic analysis of 5' strains by sequencing of the 59' end of the TEF-1α gene region revealed seven species within M. anisopliae sensu lato and two varieties outside this complex. Because initial studies on MS profiles from different cell types showed that mycelial fragments or conidia produced on nutrient-poor medium may yield too much background noise, all subsequent spectrometric analyses were performed with acidhydrolyzed conidia from 10-12 d old PDA cultures. The initial MALDI-TOF reference library included protein spectral profiles from nine taxonomically distinct, molecularly identified isolates sharing high genetic homology with the ex-type or ex-epitype isolates of these taxa in Metarhizium. A second reference library added one isolate each for M. anisopliae sensu stricto and M. robertsii. The second, larger reference library (including 11 taxa) allowed nearly perfect MALDI-TOF matching of DNA-based species identification for the 40 remaining isolates molecularly recognized as M. anisopliae sensu stricto (n = 19), M. robertsii (n = 6), M. majus (n = 3), M. lepidiotae (n = 1), M. acridum (n = 3), M. flavoviride var. pemphigi (n = 1), plus seven unidentified strains (six of them phylogenetically close to M. anisopliae sensu stricto and one outside the Metarhizium pingshaense-anisopliae-robertsii-brunneum clade). Due to the increasing frequency of phylogenetically (genomically) based taxonomic revisions of fungi, this approach is especially useful for culture collections, because once the protein profiles of Metarhizium isolates are obtained taxonomic updating of MALDI-TOF library data is easily accomplished by comparing stored profiles with those of newly proposed taxa.
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http://dx.doi.org/10.3852/13-401DOI Listing
October 2014

Evaluation of MALDI-TOF mass spectrometry for identification of environmental yeasts and development of supplementary database.

Appl Microbiol Biotechnol 2014 Jun 1;98(12):5645-54. Epub 2014 Apr 1.

Laboratório de Microbiologia Aplicada, EMBRAPA Uva e Vinho, Bento Gonçalves, RS, Brazil.

Yeast identification using traditional methods which employ morphological, physiological, and biochemical characteristics can be considered a hard task as it requires experienced microbiologists and a rigorous control in culture conditions that could implicate in different outcomes. Considering clinical or industrial applications, the fast and accurate identification of microorganisms is a crescent demand. Hence, molecular biology approaches has been extensively used and, more recently, protein profiling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proved to be an even more efficient tool for taxonomic purposes. Nonetheless, concerning to mass spectrometry, data available for the differentiation of yeast species for industrial purpose is limited and reference databases commercially available comprise almost exclusively clinical microorganisms. In this context, studies focusing on environmental isolates are required to extend the existing databases. The development of a supplementary database and the assessment of a commercial database for taxonomic identifications of environmental yeast are the aims of this study. We challenge MALDI-TOF MS to create protein profiles for 845 yeast strains isolated from grape must and 67.7 % of the strains were successfully identified according to previously available manufacturer database. The remaining 32.3 % strains were not identified due to the absence of a reference spectrum. After matching the correct taxon for these strains by using molecular biology approaches, the spectra concerning the missing species were added in a supplementary database. This new library was able to accurately predict unidentified species at first instance by MALDI-TOF MS, proving it is a powerful tool for the identification of environmental yeasts.
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http://dx.doi.org/10.1007/s00253-014-5686-7DOI Listing
June 2014

Covalent binding and anchoring of cytochrome c to mitochondrial mimetic membranes promoted by cholesterol carboxyaldehyde.

Chem Res Toxicol 2013 Oct 11;26(10):1536-44. Epub 2013 Oct 11.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo , São Paulo, SP, Brazil.

Mitochondrial cholesterol has been reported to be increased under specific pathological conditions associated with enhanced oxidative stress parameters. In this scenario, cholesterol oxidation would be increased, leading to the production of reactive aldehydes, including cholesterol carboxyaldehyde (ChAld). By using SDS micelles as a mitochondrial mimetic model, we have demonstrated that ChAld covalently modifies cytochrome c (cytc), a protein known to participate in electron transport and apoptosis signaling. This mimetic model induces changes in cytc structure in the same way as mitochondrial membranes do. Tryptic digestion of the cytc-ChAld adduct followed by MALDI-TOF/TOF analyses revealed that modifications occur at Lys residues (K22) localized at cytc site L, a site involved in protein-protein and protein-membrane interactions. Interestingly, ChAld ligation prevented cytc detachment from liposomes even under high ionic strength conditions. Overall, it can be concluded that ChAld ligation to Lys residues at site L creates a hydrophobic tail at cytc, which promotes cytc anchoring to the membrane. Although not investigated in detail in this study, cytc adduction to cholesterol derived aldehydes could have implications in cytc release from mitochondria under apoptotic stimuli.
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http://dx.doi.org/10.1021/tx4002385DOI Listing
October 2013

Conformational and functional effects induced by D- and L-amino acid epimerization on a single gene encoded peptide from the skin secretion of Hypsiboas punctatus.

PLoS One 2013 2;8(4):e59255. Epub 2013 Apr 2.

Laboratório de Espectrometria de Massa, Embrapa Recursos Genéticos e Biotecnologia, Brasília-Distrito Federal, Brasil.

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy (1)H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059255PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614549PMC
October 2013

Characterization of a novel peptide toxin from Acanthoscurria paulensis spider venom: a distinct cysteine assignment to the HWTX-II family.

Biochemistry 2013 Apr 29;52(14):2440-52. Epub 2013 Mar 29.

Laboratório de Toxinologia, Departamento de Ciências Fisiológicas, Universidade de Brasília, Brasília, DF 70910-900, Brazil.

Spider venom toxins have raised interest in prospecting new drugs and pesticides. Nevertheless, few studies are conducted with tarantula toxins, especially with species found in Brazil. This study aims to characterize chemically and biologically the first toxin isolated from Acanthoscurria paulensis venom. Ap1a consists of 48 amino acid residues and has a molecular mass of 5457.79 Da. The cloned gene encodes a putative sequence of 23 amino acid residues for the signal peptide and 27 for the pro-peptide. The sequence of the mature peptide is 60-84% identical with those of toxins of the HWTX-II family. Different from the structural pattern proposed for these toxins, the disulfide pairing of Ap1a is of the ICK type motif, which is also shared by the U1-TRTX-Bs1a toxin. Ap1a induced a dose-dependent and reversible paralytic effect in Spodoptera frugiperda caterpillars, with an ED50 of 13.0 ± 4.2 μg/g 8 h after injections. In the Drosophila melanogaster Giant Fiber circuit, Ap1a (1.14-22.82 μg/g) reduces both the amplitude and frequency of responses from GF-TTM and GF-DLM pathways, suggesting an action at the neuromuscular junction, which is mediated by glutamatergic receptors. It is also lethal to mice (1.67 μg/g, intracranial route), inducing effects similar to those reported with intracerebroventricular administration of NMDA. Ap1a (1 μM) does not alter the response induced by acetylcholine on the rhabdomyosarcoma cell preparation and shows no significant effects on hNav1.2, hNav1.4, hNav1.5, and hNav1.6 channels. Because of its unique sequence and cysteine assignment to the HWTX-II family, Ap1a is a significant contribution to the structure-function study of this family of toxins.
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http://dx.doi.org/10.1021/bi4000035DOI Listing
April 2013

Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.

PLoS One 2013 12;8(2):e56445. Epub 2013 Feb 12.

Centro de Pesquisas em Biologia Molecular e Funcional-CPBMF, Instituto Nacional de Ciência e Tecnologia em Tuberculose-INCT-TB, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Porto Alegre, Rio Grande do Sul, Brazil.

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5'-monophosphate (UMP) and pyrophosphate (PP(i)). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP(i) product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056445PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3570474PMC
August 2013

Probing protein sequences as sources for encrypted antimicrobial peptides.

PLoS One 2012 28;7(9):e45848. Epub 2012 Sep 28.

Laboratório de Espectrometria de Massa, Embrapa Recursos Genéticos e Biotecnologia, Brasília, Distrito Federal, Brazil.

Starting from the premise that a wealth of potentially biologically active peptides may lurk within proteins, we describe here a methodology to identify putative antimicrobial peptides encrypted in protein sequences. Candidate peptides were identified using a new screening procedure based on physicochemical criteria to reveal matching peptides within protein databases. Fifteen such peptides, along with a range of natural antimicrobial peptides, were examined using DSC and CD to characterize their interaction with phospholipid membranes. Principal component analysis of DSC data shows that the investigated peptides group according to their effects on the main phase transition of phospholipid vesicles, and that these effects correlate both to antimicrobial activity and to the changes in peptide secondary structure. Consequently, we have been able to identify novel antimicrobial peptides from larger proteins not hitherto associated with such activity, mimicking endogenous and/or exogenous microorganism enzymatic processing of parent proteins to smaller bioactive molecules. A biotechnological application for this methodology is explored. Soybean (Glycine max) plants, transformed to include a putative antimicrobial protein fragment encoded in its own genome were tested for tolerance against Phakopsora pachyrhizi, the causative agent of the Asian soybean rust. This procedure may represent an inventive alternative to the transgenic technology, since the genetic material to be used belongs to the host organism and not to exogenous sources.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045848PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3461044PMC
February 2013

The interaction of the antitoxin DM43 with a snake venom metalloproteinase analyzed by mass spectrometry and surface plasmon resonance.

J Mass Spectrom 2012 May;47(5):567-73

Laboratório de Espectrometria de Massa, Embrapa-Recursos Genéticos e Biotecnologia, Estação Parque Biológico, Final W5, Asa Norte, 70770-900, Brasília, DF, Brazil.

DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non-covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently neutralizes its toxicity. The aim of this study was to apply mass spectrometry (MS) and surface plasmon resonance (SPR) to improve the molecular characterization of this heterocomplex. The stoichiometry of the interaction was confirmed by nanoelectrospray ionization-quadrupole-time-of-flight MS; from native solution conditions, the complex showed a molecular mass of ~94 kDa, indicating that one molecule of jararhagin (50 kDa) interacts with one monomer of DM43 (43 kDa). Although readily observed in solution, the dimeric structure of the inhibitor was barely preserved in the gas phase. This result suggests that, in contrast to the toxin-antitoxin complex, hydrophobic interactions are the primary driving force for the inhibitor dimerization. For the real-time interaction analysis, the toxin was captured on a sensor chip derivatized with the anti-jararhagin monoclonal antibody MAJar 2. The sensorgrams obtained after successive injections of DM43 in a concentration series were globally fitted to a simple bimolecular interaction, yielding the following kinetic rates for the DM43/jararhagin interaction: k(a) = 3.54 ± 0.03 × 10(4) M(-1) s(-1) and k(d) = 1.16 ± 0.07 × 10(-5) s(-1), resulting in an equilibrium dissociation constant (K(D) ) of 0.33 ± 0.06 nM. Taken together, MS and SPR results show that DM43 binds to its target toxin with high affinity and constitute the first accurate quantitative study on the extent of the interaction between a natural inhibitor and a metalloproteinase toxin, with unequivocal implications for the use of this kind of molecule as template for the rational development of novel antivenom therapies.
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http://dx.doi.org/10.1002/jms.2990DOI Listing
May 2012

Peptidomic dissection of the skin secretion of Phasmahyla jandaia (Bokermann and Sazima, 1978) (Anura, Hylidae, Phyllomedusinae).

Toxicon 2011 Jan 12;57(1):35-52. Epub 2010 Oct 12.

Laboratório de Venenos e Toxinas Animais, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil.

The systematic investigation of the peptidic composition of the skin secretion of Phasmahyla jandaia, a phyllomedusine anuran endemic to the southern region of the Espinhaço range in Brazil, is herein reported. By means of de novo interpretation of tandem mass spectrometric data, Edman N-terminal sequencing and similarity searches, 57 peptides - including phylloseptins, dermaseptins stricto sensu, dermatoxins, hyposins, tryptophyllins, caerulein-related, bradykinin-related, bradykinin potentiating, tyrosine-rich, and opioid peptides - were sequenced. Moreover, five peptide families without significant similarity to other known molecules were verified. Differently from most Phyllomedusinae genera, the molecular diversity in the skin of representatives of Phasmahyla remained unprospected until now. Therefore, besides disclosing novel natural variants of number of bioactive peptides, the present study contributes to the understanding of the evolution of biochemical characters of the phyllomedusines.
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http://dx.doi.org/10.1016/j.toxicon.2010.09.010DOI Listing
January 2011

Cloning, purification, and partial characterization of Bacillus subtilis urate oxidase expressed in Escherichia coli.

J Biomed Biotechnol 2010 4;2010:674908. Epub 2010 Feb 4.

Laboratório de Biologia Molecular, Universidade de Brasília, 70910-900 Brasília (DF), Brazil.

Urate oxidase (EC 1.7.3.3) is an enzyme involved in purine metabolism which is used in the treatment of gout and as diagnostic reagent for detection of uric acid. In order to produce this enzyme in large quantities for biotechnological purposes, the gene coding for the Bacillus subtilis urate oxidase was cloned and heterologously expressed in Escherichia coli. Time course induction in E. coli showed an induced protein with an apparent molecular mass of approximately 60 kDa. Soluble recombinant enzyme was purified in a single-step procedure using Ni-NTA column. The enzyme was purified 2.1-fold with a yield of 56% compared to the crude extract. MALDI-TOF analysis revealed an ion with a mass of 58675 Da which is in agreement with the expected mass of the recombinant protein. The purified enzyme showed an optimal pH and temperature of 8.0 and 37 degrees C, respectively, and retained 90% of its activity after 72 hours of incubation at -20 degrees C and 4 degrees C.
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http://dx.doi.org/10.1155/2010/674908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2820260PMC
July 2010

Structural and functional analyses of Mycobacterium tuberculosis Rv3315c-encoded metal-dependent homotetrameric cytidine deaminase.

J Struct Biol 2010 Mar 24;169(3):413-23. Epub 2009 Dec 24.

Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Av. Ipiranga, 6681, Porto Alegre, RS 90619-900, Brazil.

The emergence of drug-resistant strains of Mycobacterium tuberculosis, the causative agent of tuberculosis, has exacerbated the treatment and control of this disease. Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that recycles cytidine and 2'-deoxycytidine for uridine and 2'-deoxyuridine synthesis, respectively. A probable M. tuberculosis CDA-coding sequence (cdd, Rv3315c) was cloned, sequenced, expressed in Escherichia coli BL21(DE3), and purified to homogeneity. Mass spectrometry, N-terminal amino acid sequencing, gel filtration chromatography, and metal analysis of M. tuberculosis CDA (MtCDA) were carried out. These results and multiple sequence alignment demonstrate that MtCDA is a homotetrameric Zn(2+)-dependent metalloenzyme. Steady-state kinetic measurements yielded the following parameters: K(m)=1004 microM and k(cat)=4.8s(-1) for cytidine, and K(m)=1059 microM and k(cat)=3.5s(-1) for 2'-deoxycytidine. The pH dependence of k(cat) and k(cat)/K(M) for cytidine indicate that protonation of a single ionizable group with apparent pK(a) value of 4.3 abolishes activity, and protonation of a group with pK(a) value of 4.7 reduces binding. MtCDA was crystallized and crystal diffracted at 2.0 A resolution. Analysis of the crystallographic structure indicated the presence of a Zn(2+) coordinated by three conserved cysteines and the structure exhibits the canonical cytidine deaminase fold.
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http://dx.doi.org/10.1016/j.jsb.2009.12.019DOI Listing
March 2010

Heterologous Expression and Purification of a Heat-Tolerant Staphylococcus xylosus Lipase.

Mol Biotechnol 2010 Feb;44(2):110-9

Depto de Ciência e Tecnologia de Alimentos, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil.

Staphylococcus xylosus is a microorganism involved in fermentation of meat products and also a natural producer of extracellular lipases. The aim of the present work was to clone and express in E. coli a lipase from S. xylosus (AF208229). This lipase gene (1084 bp) was amplified from a S. xylosus strain isolated from naturally fermented salami and introduced in pET14b expression vector in order to express the recombinant fusion protein (histidine-tagged lipase) in E. coli. Recombinant histidine-tagged S. xylosus lipase was purified by affinity chromatography in an HPLC system. The histidine-tagged lipase is a monomer in solution, as determined by size-exclusion chromatography. It presents a high lipase activity at pH 9.0 and 42 degrees C for p-nitrophenyl acetate and p-nitrophenyl butyrate, among seven different esters assayed (pNPC(2), pNPC(4), pNPC(10), pNPC(12), pNPC(14), pNPC(16), pNPC(18)). Moreover, the enzyme presented a quite interesting thermal stability, after an incubation period of 10 min at 95 degrees C, 77% of the initial activity was retained.
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http://dx.doi.org/10.1007/s12033-009-9218-0DOI Listing
February 2010

Leptodactylus ocellatus (Amphibia): mechanism of defense in the skin and molecular phylogenetic relationships.

J Exp Zool A Ecol Genet Physiol 2010 Jan;313(1):1-8

Núcleo de Pesquisa em Biodiversidade e Biotecnologia, Campus Ministro Reis Velloso (CMRV), Universidade Federal do Piauí-UFPI, Parnaíba, Piauí, PI, Brazil.

Amphibian antimicrobial peptides have been known for many decades and several of them have already been isolated. However, the number of species investigated is still small. Herein, we report on the skin secretions of Leptodactylus ocellatus, which were extracted by mild electrical stimulation and its semi-preparative reverse-phase chromatography was resolved in more than 30 fractions. Among these fractions, two novel antimicrobial peptides were isolated and their amino acid sequences determined by de novo sequencing. The ocellatins-5 and -6 (21 and 22 amino acid residues, respectively) are amidated at the C-terminus. Ocellatins inhibited the growth of reference strains of both Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) with minimal inhibition concentration values in the range of 32-128 microg/mL. The amino acid sequence of the peptides shows structural similarity with members of the antimicrobial peptides found in the skin secretion of other leptodactylid frogs. This observation is consistent with the hypothesis that many frog skin antimicrobial peptides are related evolutionarily, having arisen from multiple duplications of an ancestral gene that existed before the radiation of the different species.
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http://dx.doi.org/10.1002/jez.551DOI Listing
January 2010

A novel antimicrobial peptide from Crotalaria pallida seeds with activity against human and phytopathogens.

Curr Microbiol 2009 Oct 30;59(4):400-4. Epub 2009 Jul 30.

Universidade Católica de Brasília, Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Brasília, DF, Brazil.

An actual severe problem in agriculture consists of an expressive increase of economical losses caused by fungi and resistant bacteria toward antibiotics. In order to find a solution to this problem, several studies have been concentrating on the screening of novel plant defense peptides with antimicrobial activities. These peptides are commonly characterized by having low molecular masses and cationic charges. The present work reports the purification and characterization of a novel plant peptide with molecular mass of 5340 Da, named Cp-AMP, from seeds of C. pallida, a typical plant from Caatinga biome. Purification was achieved using a size exclusion S-200 column followed by reversed-phase chromatography on Vydac C18-TP column. In vitro assays indicated that Cp-AMP was able to inhibit the development of filamentous fungi Fusarium oxysporum as well as the gram-negative bacterium Proteus sp. The identification of Cp-AMP could contribute, in the near future, to the development of biotechnological products, such as transgenic plants with enhanced resistance to pathogenic fungi and/or of antibiotics production derived from plant sources in order to control bacterial infections.
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http://dx.doi.org/10.1007/s00284-009-9451-6DOI Listing
October 2009

Antiplasmodial and antileishmanial activities of phylloseptin-1, an antimicrobial peptide from the skin secretion of Phyllomedusa azurea (Amphibia).

Exp Parasitol 2009 Sep 19;123(1):11-6. Epub 2009 May 19.

Laboratório de Imunologia Celular, Area de Patologia, Faculdade de Medicina, Universidade de Brasília, 70910-900 Brasília, DF, Brazil.

The development of drug resistance by infectious agents represents a major hindrance for controlling parasitic diseases and has stimulated the search for new compounds. We have previously shown that phylloseptin-1 (PS-1), a cationic peptide from the skin secretion of Phyllomedusa azurea, exhibited potent antimicrobial activity. Now we evaluate the effect of PS-1 on Leishmania amazonensis and Plasmodium falciparum. Concentrations as low as 0.5 microg/mL of PS-1 exhibited antileishmanial activity comparable to that of antimoniate of N-metilglucamine, while the antiplasmodial effect of PS-1 was evident at the concentration of 16 microg/mL, and reached an activity comparable to that of artesunate, at the concentration of 64 microg/mL. The high antiparasitic activity of PS-1, together with the unrelatedness of its chemical structure to any present antimicrobial drug, which prevents the development of cross-resistance, together with its non-toxicity to mammalian cells make this peptide a promising candidate for the treatment of malaria and leishmaniasis.
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http://dx.doi.org/10.1016/j.exppara.2009.05.002DOI Listing
September 2009

Post-secretory events alter the peptide content of the skin secretion of Hypsiboas raniceps.

Biochem Biophys Res Commun 2008 Dec 29;377(4):1057-61. Epub 2008 Oct 29.

EMBRAPA Recursos Genéticos e Biotecnologia, Parque Estação Biológica, 70700-900 Brasília, DF, Brazil.

A novel family of antimicrobial peptides, named raniseptins, has been characterized from the skin secretion of the anuran Hypsiboas raniceps. Nine cDNA molecules have been successfully cloned, sequenced, and their respective polypeptides were characterized by mass spectrometry and Edman degradation. The encoded precursors share structural similarities with the dermaseptin prepropeptides from the Phyllomedusinae subfamily and the mature 28-29 residue long peptides undergo further proteolytic cleavage in the crude secretion yielding consistent fragments of 14-15 residues. The biological assays performed demonstrated that the Rsp-1 peptide has antimicrobial activity against different bacterial strains without significant lytic effect against human erythrocytes, whereas the peptide fragments generated by endoproteolysis show limited antibiotic potency. MALDI imaging mass spectrometry in situ studies have demonstrated that the mature raniseptin peptides are in fact secreted as intact molecules within a defined glandular domain of the dorsal skin, challenging the physiological role of the observed raniseptin fragments, identified only as part of the crude secretion. In this sense, stored and secreted antimicrobial peptides may confer distinct protective roles to the frog.
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http://dx.doi.org/10.1016/j.bbrc.2008.10.102DOI Listing
December 2008
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