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Abstract: Cotton (Gossypium hirsutum L.) is an important agricultural commodity, which is attacked by several pests such as the cotton boll weevil Anthonomus grandis. Adult A. grandis feed on fruits and leaf petioles, reducing drastically the crop production. The predominance of boll weevil digestive serine proteinases has motivated inhibitor screenings in order to discover new ones with the capability to reduce the digestion process. The present study describes a novel proteinase inhibitor from chickpea seeds (Cicer arietinum L.) and its effects against A. grandis. This inhibitor, named CaTI, was purified by using affinity Red-Sepharose Cl-6B chromatography, followed by reversed-phase HPLC (Vydac C18-TP). SDS-PAGE and MALDI-TOF analyses, showed a unique monomeric protein with a mass of 12,877 Da. Purified CaTI showed significant inhibitory activity against larval cotton boll weevil serine proteinases (78%) and against bovine pancreatic trypsin (73%), when analyzed by fluorimetric assays. Although the molecular mass of CaTI corresponded to α-amylase/trypsin bifunctional inhibitors masses, no inhibitory activity against insect and mammalian α-amylases was observed. In order to observe CaTI in vivo effects, an inhibitor rich fraction was added to an artificial diet at different concentrations. At 1.5% (w/w), CaTI caused severe development delay, several deformities and a mortality rate of approximately 45%. These results suggested that CaTI could be useful in the production of transgenic cotton plants with enhanced resistance toward cotton boll weevil. [Copyright &y& Elsevier]

The present work reports the isolation, characterization and the complete sequence of a phospholipase A 2 (PLA 2) 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 2 activity was purified, assayed, sequenced and named Pa-PLA 2. The Pithecopus azureus skin phospholipase A 2 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 2 enzymatic activity was qualitatively evaluated and compared to classical viperid PLA 2 showing that both, native and deglycosylated Pa-PLA 2 forms, are catalytically functional. The tridimensional molecular model of Pa-PLA 2 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 2 catalytic pocket and its substrates. • Complete sequence of a phospholipase A 2 from the skin secretion of Pithecopus azureus. • Two glycosylation sites in the PLA 2 polypeptide chain (N67 and N108). • Complex mannose-rich glycans at both N-glycosylation sites. [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • A dual function whey ingredient (PC3) was developed and 21 peptides were identified. • PC3 hydrolysate showed high vascular relaxation (65%) in an ex-vivo model. • PC3 hydrolysate showed high solubility in a wide pH range (1–12). • The hydrolysate obtained presented great oil absorption capacity. • Sensory analysis showed a high acceptance of the ingredient among 100 participants. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: Three dermaseptins, DS 01, DD K, and DD L, were compared with respect to their structural features and interactions with liposomes. Circular dichroic spectra at alcohols of different chain lengths revealed that DS 01 has the higher helicogenic potential in hydrophobic media. Binding of DS 01, DD K, and DD L to liposomes induced significant blue shifts of the emission spectra of the single tryptophan located at position 3 of all sequences indicating association of the peptides with bilayers. Kinetics evaluation of atomic force microscopy images evidenced the strong fusogenic activity of DS 01 whereas DD K and DD L showed increased lytic activities. [Copyright &y& Elsevier]

Abstract: The present study reports the structural characteristics, the biological activities, and preliminary clinical investigations of three synthetic members of the dermaseptin family of antimicrobial peptides. The three peptides showed similar tendencies to form α-helical structures in non-polar media. The antimicrobial activity towards bacteria and fungi was determined in the micromolar concentration and the peptides did not influenced peritoneal cells viability. One of the peptides was intravenously administered in mice at concentrations similar to those of antibiotics employed in bacterial/fungal infections and it did not cause any detectable changes in cells and tissues. [Copyright &y& Elsevier]

Abstract: This communication reports the separation of 80 fractions from the venom of the Ischnuridae scorpion Opisthacanthus cayaporum by high-performance liquid chromatography (HPLC). From these, 93 distinct components were identified by liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) analysis, with molecular weights varying from 229.2 to 61,144.0 atomic mass units. Additionally, the HPLC fractions were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) which resulted in 221 distinct components, among which were 52 of the 93 obtained by LC/ESI-MS. The entire set of different molecular species found (total of 262 molecular masses) has a trimodal molecular weight distribution, with 42% of the components possessing 229.2–2985.3Da, 37% within the range of 3045.0–7258.6Da and 12% within the range 7458.4–9429Da. Seventeen peptides/proteins were isolated and were sequenced by Edman degradation, among which were a scorpine-like peptide (8315Da), presenting antimicrobial activity, and two phospholipase A2 with a molecular weight around 14kDa. The pharmacological effects of the venom were tested on isolated rat and insect (cockroach) nerves using the single sucrose-gap assay. The ED50 of the venom was 1.1mg/ml in insect nerves. Venom concentrations in the order of 3mg/ml causes only 9% reduction of compound action potentials (APs) of rat nerves, suggesting that this venom is rather specific for insects. Comparative analysis of venom from male and female O. cayaporum was performed by HPLC and MALDI-TOF-MS showing no qualitative variations, but rather quantitative differences among both samples. [Copyright &y& Elsevier]

Abstract: DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43mol% (2mmolL−1) than in its absence (93μmolL−1). Apparent binding constant values were 2160 and 10,000Lmol−1, respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide. [Copyright &y& Elsevier]

Abstract: Antimicrobial peptides are considered part of the innate immune system of the majority of living organisms. Most of these molecules are small, cationic and show amphiphilic nature. The skin secretions of Leptodactylus syphax were extracted by mild electrical stimulation and its semipreparative reverse-phase chromatography was resolved in more than 40 fractions. Among these fractions, an antimicrobial peptide was isolated and its amino acid sequence determined by de novo sequencing. Six other truncated forms were characterized in skin secretion. The longest one (25 amino acid residues), named syphaxin (SPX), is amidated at the C-terminal, and shares strong sequence similarity with antimicrobial peptides found in the skin secretion of leptodactylid frogs. Two of the truncated peptides (SPX(1–22) and SPX(1–16)) were tested against Escherichia coli and Staphylococcus aureus, showing low minimal inhibitory concentration (MIC) and no significant toxicity towards blood cells, including both leukocytes and erythrocytes, based on their direct incubation in whole blood at the highest MIC concentration (64μg/mL). [Copyright &y& Elsevier]

Abstract: Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish. By using four different techniques—mouse bioassay, ELISA, HPLC and mass spectrometry—we evaluated the toxicity in the extracts of C. asellus, a freshwater puffer fish from the rivers of the Amazon, and identified for the first time the components responsible for its toxicity. The T20G10 monoclonal antibody raised against TTX, and employed in an indirect competitive enzyme immunoassay, showed very low affinity for the C. asellus extracts, indicating that TTX and its analogs are not the main toxic components of the extracts. This antibody was efficient in detecting presence of TTX in a total extract of Sphoeroides spengleri, which is one of the most toxic puffer fish found in the Atlantic coast. Extracts of C. asellus were toxic when administered intraperitonially into mice with an average toxicity of 38.6±12 mouse unit (MU)/g, while HPLC analysis indicated a lower toxin content (7.6±0 5MU/g). The HPLC profile showed no traces of TTX, but only the presence of PSPs (STX, GTX 2 and GTX 3). These toxins were also confirmed by electrospray ionization mass spectrometry. [Copyright &y& Elsevier]

Abstract: The increase in the emergence of antibiotic-resistant microorganisms and difficult to treat infections caused by these pathogens stimulate research aiming the identification of novel antimicrobials. Skin secretion of amphibian contains a large number of biologically active compounds, including compounds that performance defense mechanisms against microorganisms. In the present work, two antimicrobial bufadienolides, telocinobufagin (402.1609Da) and marinobufagin (400.1515Da), were isolated from skin secretions of the Brazilian toad Bufo rubescens. The specimens were collected in Brasilia (Distrito Federal, Brazil), the skin secretions extracted by electric stimulation, and submitted to purification by RP-HPLC. The molecular structure and mass determination were done by 1H and 13C NMR and mass spectrometry data, respectively. The antimicrobial activity was performed by liquid growth inhibition against Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentrations of telocinobufagin and marinobufagin were, respectively, 64.0 and 16.0μg/mL for E. coli and both 128μg/mL for S. aureus. Besides the antimicrobial activity both bufadienolides promoted an increase of the contraction force in isolated frog ventricle strips. [Copyright &y& Elsevier]

Abstract: Six novel peptides called phylloseptins (PS-1, -2, -3, -4, -5, and -6) showing anti-bacterial (PS-1) and anti-protozoan (PS-4 and -5) activities were isolated from the skin secretion of the Brazilian tree-frogs, Phyllomedusa hypochondrialis and Phyllomedusa oreades. Phylloseptins have a primary structure consisting of 19–21 amino acid residues (1.7–2.1kDa). They have common structural features, such as a highly conserved N-terminal region and C-terminal amidation. Phylloseptin-1 (FLSLIPHAINAVSAIAKHN-NH2) demonstrated a strong effect against Gram-positive and Gram-negative bacteria (MICs ranging from 3 to 7.9μM), without showing significant hemolytic activity (<0.6% at the MIC range) towards mammalian cells. Atomic force microscopy experiments indicated that the bacteriolytic properties of these peptides might be related to their disruptive action on the cell membrane, characterized by a number of bubble-like formations, preceding every cell lysis. PS-4 and PS-5 showed anti-protozoan activity with IC50 at about 5μM for Trypanossoma cruzi. [Copyright &y& Elsevier]

Abstract: Tetrodotoxin (TTX) is one of the most potent toxin already isolated, which occurs in a wide range of marine as well as terrestrial animals such as in newts and anurans. In this work, the occurrence of TTX and analogues was examined in three brachycephalid species: Brachycephalus ephippium, B. nodoterga and B. pernix using LC-FLD and LC-MS/MS. In toxicity assay (intra-peritonial injection in mice) B. nodoterga extracts were non-toxic, while B. pernix extract exhibit the highest toxicity among the studied species. Skin showed the highest toxic, followed by the liver. Retention time data in the LC-FLD system indicated the presence of TTX, 4-epiTTX, 4,9-anhydroTTX and TDA, SIM data confirmed the presence of these compounds and revealed other analogs such as 11-norTTX-6(S)-ol, 5-deoxyTTX, 11-deoxyTTX, 11-oxoTTX, 6-epiTTX. Two new components were also identified by mass spectrometry (348 and 330Da). These unknown compounds have daughter ions similar to TTX, suggesting new putative TTX analogues. [Copyright &y& Elsevier]

Two isoforms of a thrombin-like enzyme designated TLE-B and TLE-P were purified from the venoms of Lachesis muta muta (bushmaster) snakes captured in two different geographical localities, Manaus (Brazil) and Pucallpa (Peru´). TLE-B and TLE-P showed Mr values of 44 000 and 43 000 under reducing conditions on SDS-PAGE, which decreased to 27 000 after deglycosylation with N-glycosidase F (PNGase F). The purified proteinases split off fibrinopeptide A rapidly from human fibrinogen and fibrinopeptide B more slowly. In addition, both enzymes released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bβ-chain. Their specific clotting activities were equivalent to 1000 and 900 NIH thrombin units/mg on human fibrinogen and 526 and 606 NIH thrombin units/mg on bovine fibrinogen for TLE-B and TLE-P, respectively. Kinetic properties of these enzymes were determined using representative chromogenic substrates. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Purified rabbit IgG against TLE-B reacted with both enzymes forming a continuous precipitin line on immunodiffusion. Furthermore, Western blot and indirect ELISA were used to compare the antigenic cross-reactivity for both enzymes as well as the venoms of L. muta muta and Bothrops snakes. Incubation of human α2-macroglobulin (α2-M) with each enzyme at molar ratios of 1:1, 1:2 and 1:4 enzyme:inhibitor resulted in retarding their clotting activities by approximately 12 times, whereas their amidolytic activities were not affected. However, the Mr 180 000 subunits of α2-M were not cleaved by these enzymes, suggesting that α2-M inhibits TLEs by steric hindrance. Similarly, inhibitions of their clotting activities were obtained using high concentrations of rabbit IgG (40 μg, corresponding to molar ratio enzyme:inhibitor of 1:2) against TLE-B. [Copyright &y& Elsevier]