No abstract

The occurrence and spatial distribution of the neuropeptides AmTRP-5 and AST-1 in the honeybee brain were monitored via MALDI spectral imaging according to the ontogeny of Africanized Apis mellifera. The levels of these peptides increased in the brains of 0-15 day old honeybees, and this increase was accompanied by an increase in the number of in-hive activities performed by the nurse bees, followed by a decrease in the period from 15 to 25 days of age, in which the workers began to perform activities outside the nest (guarding and foraging). The results obtained in the present investigation suggest that AmTRP-5 acts in the upper region of both pedunculi of young workers, possibly regulating the cell cleaning and brood capping activities. Meanwhile, the localized occurrence of AmTRP-5 and AST-1 in the antennal lobes, subesophageal ganglion, upper region of the medulla, both lobula, and α- and β-lobes of both brain hemispheres in 20 to 25 day old workers suggest that the action of both neuropeptides in these regions may be related to their localized actions in these regions, regulating foraging and guarding activities. Thus, these neuropeptides appear to have some functions in the honeybee brain that are specifically related to the age-related division of labor.

Polycationic peptides may present their C-termini in either amidated or acidic form; however, the effects of these conformations on the mechanisms of interaction with the membranes in general were not properly investigated up to now. Protonectarina-MP mastoparan with an either amidated or acidic C-terminus was utilized to study their interactions with anionic and zwitterionic vesicles, using measurements of dye leakage and a combination of H/D exchange and mass spectrometry to monitor peptide-membrane interactions. Mast cell degranulation, hemolysis and antibiosis assays were also performed using these peptides, and the results were correlated with the structural properties of the peptides. The C-terminal amidation promotes the stabilization of the secondary structure of the peptide, with a relatively high content of helical conformations, permitting a deeper interaction with the phospholipid constituents of animal and bacterial cell membranes. The results suggested that at low concentrations Protonectarina-MP interacts with the membranes in a way that both terminal regions remain positioned outside the external surface of the membrane, while the α-carbon backbone becomes partially embedded in the membrane core and changing constantly the conformation, and causing membrane destabilization. The amidation of the C-terminal residue appears to be responsible for the stabilization of the peptide conformation in a secondary structure that is richer in α-helix content than its acidic congener. The helical, amphipathic conformation, in turn, allows a deeper peptide-membrane interaction, favoring both biological activities that depend on peptide structure recognition by the GPCRs (such as exocytosis) and those activities dependent on membrane perturbation (such as hemolysis and antibiosis).

No abstract

Previous studies of the fractionated venom of the Brazilian armed spider Phoneutria nigriventer, obtained by gel filtration, have demonstrated the presence of a fraction PhM, a pool of small peptides (up to 2000 Da) that provoke contractions in smooth muscle of guinea pig ileum. Initial attempts to sequence these peptides were largely unsuccessful because of the low purification yield and the fact that the majority seemed to be blocked at their N-termini. In the present work, analysis of this venom fraction by mass spectrometry has revealed the existence of a highly complex mixture of peptides with molecular weights corresponding to those observed for the muscle-active peptides previously described (800-1800 Da). These peptides appear to be a family of isoforms with some particular features. The amino acid sequences of 15 isoforms have been determined by tandem mass spectrometry (MS/MS) using both electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q/ToFMS) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-ToF/ToFMS). These molecules contain post-translational modifications such as proteolysis and C-terminal amidation, which combine to generate additional isoforms. All the isoforms sequenced in this study possess an N-terminal pyroglutamic acid residue. A search for sequence similarities with other peptides in databanks revealed that these peptides are structurally related to the tachykinins, a family of neuro-hormone peptides. The data obtained in this study will be essential for the subsequent steps of this research, the synthesis of these peptides and pharmacological characterization of their biological activity.

In this study, a series of mastoparan analogs were engineered based on the strategies of Ala and Lys scanning in relation to the sequences of classical mastoparans. Ten analog mastoparans, presenting from zero to six Lys residues in their sequences were synthesized and assayed for some typical biological activities for this group of peptide: mast cell degranulation, hemolysis, and antibiosis. In relation to mast cell degranulation, the apparent structural requirement to optimize this activity was the existence of one or two Lys residues at positions 8 and/or 9. In relation to hemolysis, one structural feature that strongly correlated with the potency of this activity was the number of amino acid residues from the C-terminus of each peptide continuously embedded into the zwitterionic membrane of erythrocytes-mimicking liposomes, probably due to the contribution of this structural feature to the membrane perturbation. The antibiotic activity of mastoparan analogs was directly dependent on the apparent extension of their hydrophilic surface, i.e., their molecules must have from four to six Lys residues between positions 4 and 11 of the peptide chain to achieve activities comparable to or higher than the reference antibiotic compounds. The optimization of the antibacterial activity of the mastoparans must consider Lys residues at the positions 4, 5, 7, 8, 9, and 11 of the tetradecapeptide chain, with the other positions occupied by hydrophobic residues, and with the C-terminal residue in the amidated form. These requirements resulted in highly active AMPs with greatly reduced (or no) hemolytic and mast cell degranulating activities.

Invasive fungal infections, such as cryptococcosis and paracoccidioidomycosis are associated with significant rates of morbidity and mortality. Cryptococcosis, caused by Cryptococcus neoformans, is distributed worldwide and has received much attention as a common complication in patients with HIV. Invasive fungal infections are usually treated with a combination of amphotericin B and azoles. In addition, 5-fluorocytosine (5-FC) is applied in cryptococcosis, specifically to treat central nervous system infection. However, host toxicity, high cost, emerging number of resistant strains, and difficulty in developing new selective antifungals pose challenges. The need for new antifungals has therefore prompted a screen for inhibitory peptides, which have multiple mechanisms of action. The honeycomb moth Galleria mellonella has been widely used as a model system for evaluating efficacy of antifungal agents. In this study, a peptide analog from the mastoparan class of wasps (MK58911) was tested against Cryptococcus spp. and Paracoccidioides spp. In addition, peptide toxicity tests on lung fibroblasts (MRC5) and glioblastoma cells (U87) were performed. Subsequent tests related to drug interaction and mechanism of action were also performed, and efficacy and toxicity of the peptide were evaluated in vivo using the G. mellonella model. Our results reveal promising activity of the peptide, with an MIC in the range of 7.8–31.2 μg/mL, and low toxicity in MRC and U87 cells (IC50 > 500 μg/mL). Taken together, these results demonstrate that MK58911 is highly toxic in fungal cells, but not mammalian cells (SI > 16). The mechanism of toxicity involved disruption of the plasma membrane, leading to death of the fungus mainly by necrosis. In addition, no interaction with the drugs amphotericin B and fluconazole was found either in vitro or in vivo. Finally, the peptide showed no toxic effects on G. mellonella, and significantly enhanced survival rates of larvae infected with C. neoformans. Although not statistically significant, treatment of larvae with all doses of MK58911 showed a similar trend in decreasing the fungal burden of larvae. These effects were independent of any immunomodulatory activity. Overall, these results present a peptide with potential for use as a new antifungal drug to treat systemic mycoses.

RESUMO -O experimento foi conduzido com os objetivos de avaliar o efeito da suplementação de fitase em dietas sobre a energia metabolizável (EMAn) e sobre os coeficientes de digestibilidade ileal aparente de matéria seca, proteína bruta, cálcio e fósforo e determinar a deposição de cinzas, cálcio e fósforo na tíbia de frangos de corte. Foram utilizados 350 frangos de corte machos Ross de 16 a 25 dias de idade, distribuídos num delineamento inteiramente ao acaso, com cinco dietas e dez repetições de sete aves por unidade experimental. As dietas, à base de milho e de farelo de soja, foram formuladas considerando a disponibilidade de energia metabolizável, proteína bruta, lisina, cálcio e fósforo disponível, de acordo com a matriz nutricional da enzima fitase, e suplementadas com 0,5% de oxido crômico. As dietas foram: controle positivo; controle negativo 1; controle negativo 2; controle negativo 1 + 250 uft; controle negativo 2 + 500 uft. A suplementação de enzima fitase nos níveis de 250 e de 500 uft/kg de dieta melhorou os valores energéticos das dietas, que aumentaram, em média, 36 e 54 kcal/kg de MS, respectivamente. A utilização de fitase melhorou os coeficientes de digestibilidade da proteína bruta e do fósforo, cujos maiores valores foram obtidos com a suplementação de 500 uft/kg de ração. A suplementação de fitase melhorou o coeficiente de digestibilidade e a retenção de fósforo, reduzindo o fósforo excretado, e aumentando a composição de fósforo na tíbia das aves, principalmente nos frangos de corte alimentados com a dieta suplementada com fitase (500 uft/kg). A suplementação de fitase (500 uft/kg) melhora o coeficiente de digestibilidade da proteína bruta e do fósforo, melhora a retenção de fósforo e diminui a excreção de fósforo de frangos de corte.Palavras-chave: cálcio, digestibilidade, enzima, fósforo, tíbia Phytase dietetic supplementation on nutrients metabolism of broilers ABSTRACT -The experiment was carried out to evaluate the effect of phytase enzyme supplementation in diets on the metabolizable energy (ME) and on the apparent ileum digestibility coefficients of the dry matter, crude protein, calcium and phosphorous, and to determine the deposition of ashes, calcium and phosphorous on the tibia of broilers.It was used 350 Ross male broilers from 16 to 25 days of age distributed in a complete randomized design with five diets and ten replicates of seven birds per each experimental unit. The diets which were corn and soybean meal based were formulated considering the availability of metabolizable energy, crude protein, lysine, calcium, and available phosphorus according to the nutritional matrix of phytase enzyme, and supplemented with 0.5% of chromium oxide. The diets were the following: positive control; negative control 1; negative control 2; negative control 1 + 250 ftu; negative control 2 + 500 ftu. The phytase enzyme supplementation at levels of 250 and 500 ftu/kg of diet improved the energetic values of the diets by 36 and 54 kcal/kg of dry matter, respectively. The use of phytase improved...