Mass spectrometric characterization of two novel inflammatory peptides from the venom of the social wasp Polybia paulista

The social wasp P. paulista is relatively common in southeast Brazil causing many medically important stinging incidents. The seriousness of these incidents is dependent on the amount of venom inoculated by the wasps into the victims, and the characteristic envenomation symptoms are strongly dependent on the types of peptides present in the venom. In order to identify some of these naturally occurring peptides available in very low amounts, an analytical protocol was developed that uses a combination of reversed-phase and normal-phase high-performance liquid chromatography (HPLC) of wasp venom for peptide purification, with matrix-assisted laser desorption/ionization time-of-flight post-source decay mass spectrometry (MALDI-Tof-PSD-MS) and low-energy collision-induced dissociation (CID) in a quadrupole time-of-flight tandem mass spectrometry (QTof-MS/MS) instrument for peptide sequencing at the sub-picomole level. The distinction between Leu and Ile was achieved both by observing d-type fragment ions obtained under CID conditions and by comparison of retention times of the natural peptides and their synthetic counterparts (with different combinations of I and/or L at N- and C-terminal positions). To distinguish the isobaric residues K and Q, acetylation of peptides was followed by Q-Tof-MS analysis. The primary sequences obtained were INWLKLGKMVIDAL-NH2 (MW 1611.98Da) and IDWLKLGKMVMDVL-NH2 (MW 1658.98Da). Micro-scale bioassay protocols characterized both peptides as presenting potent hemolytic action, mast cell degranulation, and chemotaxis of poly-morphonucleated leukocyte (PMNL) cells. The primary sequences and the bioassay results suggest that these toxins constitute members of a new sub-class of mastoparan toxins, directly involved in the occurrence of inflammatory processes after wasp stinging. Copyright (C) 2004 John Wiley Sons, Ltd.

Advantages of using nested collision induced dissociation/post-source decay with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: sequencing of novel peptides from wasp venom

We have examined the applicability of the 'nested' collision induced dissociation/post-source decay (CID/PSD) method to the sequencing of novel peptides from solitary wasps which have neurotoxic venom for paralyzing other insects. The CID/PSD spectrum of a ladder peptide derived from an exopeptidase digest was compared with that of the intact peptide. The mass peaks observed only in the CID/PSD spectrum of a ladder peptide were extracted as C-terminal fragment ions. Assignment of C-terminal fragment ions enabled calculation of N-terminal fragment masses, leading to differentiation between N-terminal fragment ions and internal fragment ions. This methodology allowed rapid and sensitive identification by removing ambiguity in the assignment of the fragment ions, and proved useful for sequencing unknown peptides, in particular those available as natural products with a limited supply. Copyright (C) 2000 John Wiley & Sons, Ltd.

Application of the comet assay in erythrocytes of Oreochromis niloticus (Pisces): a methodological comparison

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effect of topical application of juvenile hormone (JH) in honeybee worker larvae on the development of the Dufour's and Koschewnikow's glands

In order to investigate the action of the juvenile hormone (JH) on honeybee caste differentiation two exocrine glands, Koschewnikow and Dufour glands, were chosen for study. Two combs (I & II) were taken from a single posture of a queen to use for this research. In comb I the larvae were treated with a topical application of JH in Acetone, and those from the comb II (control group) received only Acetone. Immediately after the emergence of the workers, their glands were dissected and prepared for microscopic measurements. The results showed cell area reduction in the Koschewnikow gland induced by the JH application. The results for the Dufour gland displayed taller epithelial cells with the JH application. The difference in glandular responses to the JH relates to gland function, hormone targets, and individual homeostasis.

Adaptation of the reservoir fish assemblage index (RFAI) for assessing the Barra Bonita Reservoir (São Paulo, Brazil)

In order to evaluate the environmental condition of the Barra Bonita Reservoir, we adapted the Index of Biotic Integrity (IBI). We chose 24 sampling sites in which three types of habitats were sampled: the mouths of tributaries, the central reservoir and the lateral reservoir. Fish were caught in two seasons (dry and rainy) using 10 gillnet gangs, with meshes ranging from 3 to 12 cm between opposite knots, and funnel traps. Abiotic and biotic variables were measured. Due to the artificial nature of the reservoir, the term biotic integrity was considered inappropriate and the term RFAI (Reservoir Fish Assemblage Index) was adopted. Twelve metrics from sixteen possibles were selected using the Pearson correlation coefficient. The reference conditions were set up based on the criteria of the best condition observed. For each metric, a score of 1, 3 or 5 was assigned as it strongly departs (1), slightly departs (3) or approaches (5) the reference condition. The index value is the sum of the metrics partial scores and ranges from 12 to 60. To evaluate the importance of the unit of measurement of the metric, the index was calculated in fish number (RFAI(N)) and in weight (RFAI(w)). The correlation between RFAIN and RFAIw was very high (r = 0.90, n = 46) indicating that the unit of measurement does not influence the final result of the index. Most of the sampling sites were classified in the 'reasonable' RFAI category. Only the central sites were classified as 'poor'. To validate the RFAI, another index, the Habitat Quality Index (HQI), was built starting from the physiochemical and habitat variables collected. The correlation of the RFAI with the HQI was highly significant (RFAIN, r = 0.37; RFAI(w), r = 0.47; n = 46), indicating that they respond in the same way to environmental degradation. The HQI metrics which most affect RFAI were depth, surrounding landscape and macrophyte presence/absence. Copyright C) 2007 John Wiley & Sons, Ltd.

Growth factors and glucose homeostasis in diabetic rats: effects of exercise training

To investigate the alterations of glucose homeostasis and variables of the insulin-like growth factor-I (IGF- 1) growth system in sedentary and trained diabetic (TD) rats, Wistar rats were divided into sedentary control (SC), trained control (TC), sedentary diabetic (SD), and TD groups. Diabetes was induced by Alloxan (35 mg kg(-1) b.w.). Training program consisted of swimming 5 days week(-1), 1 h day(-1), during 8 weeks. Rats were sacrificed and blood was collected for determinations of serum glucose, insulin, growth hormone (GH), IGF-1, and IGF binding protein-3(IGFBP-3). Muscle and liver were removed to evaluate glycogen content. Cerebellum was extracted to determinate IGF-1 content. Diabetes decreased serum GH, IGF-1, IGFBP-3, liver glycogen, and cerebellum IGF-1 peptide content in baseline condition. Physical training recovered liver glycogen and increased serum and cerebellum IGF-1 peptide in diabetic rats. Physical training induces important metabolic and hormonal alterations that are associated with an improvement in glucose homeostasis and serum and cerebellum IGF-1 concentrations. Copyright (C) 2009 John Wiley & Sons, Ltd.

Determination of the lactate threshold and maximal blood lactate steady state intensity in aged rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Changes of glycogen content in liver, skeletal muscle, and heart from fasted rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Antioxidant potential of oregano (Oreganum vulgare L.), basil (Ocimum basilicum L.) and thyme (Thymus vulgaris L.): application of oleoresins in vegetable oil

Por razões econômicas e de proteção à saúde, pesquisas têm sido dirigidas para ampliar a estabilidade dos óleos vegetais. Existe uma tendência para a adição de antioxidantes naturais, em particular, um crescente interesse em ervas e especiarias. Desta forma, os objetivos deste trabalho foram avaliar o potencial antioxidante das oleorresinas de orégano, manjericão e tomilho e seu comportamento ao serem aplicadas ao óleo de soja em diferentes concentrações. Numa primeira etapa foi determinada a atividade antioxidante pelo sistema β-caroteno/ácido linoleico e a quantificação de compostos fenólicos totais. Posteriormente, foram adicionadas ao óleo de soja diferentes concentrações das oleorresinas (500 a 3000 mg.kg-1) e analisado seu potencial antioxidante por meio da estabilidade oxidativa utilizando o Rancimat. A concentração de 3000 mg.kg-1das oleorresinas de orégano e tomilho foi a que apresentou melhor estabilidade oxidativa ao óleo de soja, o que as tornam alternativa natural na conservação de óleos vegetais.

Application of abrupt cut-off models in the analysis of the capacitance spectra of conjugated polymer devices

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Study of the mechanism of action of anoplin, a helical antimicrobial decapeptide with ion channel-like activity, and the role of the amidated C-terminus

Anoplin, an antimicrobial, helical decapeptide from wasp venom, looses its biological activities by mere deamidation of its C-terminus. Secondary structure determination, by circular dichroism spectroscopy in amphipathic environments, and lytic activity in zwitterionic and anionic vesicles showed quite similar results for the amidated and the carboxylated forms of the peptide. The deamidation of the C-terminus introduced a negative charge at an all-positive charged peptide, causing a loss of amphipathicity, as indicated by molecular dynamics simulations in TFE/water mixtures and this subtle modification in a peptide's primary structure disturbed the interaction with bilayers and biological membranes. Although being poorly lytic, the amidated form, but not the carboxylated, presented ion channel-like activity on anionic bilayers with a well-defined conductance step; at approximately the same concentration it showed antimicrobial activity. The pores remain open at trans-negative potentials, preferentially conducting cations, and this situation is equivalent to the interaction of the peptide with bacterial membranes that also maintain a high negative potential inside. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Application of crude xylanase from Bacillus licheniformis 77-2 to the bleaching of eucalyptus Kraft pulp

Alkalophilic Bacillus licheniformis 77-2 produced an extracellular alkali-tolerant xylanase with negligible cellulase activity in medium containing corn straw. The effectiveness of crude xylanase on treatment of eucalyptus Kraft pulp was evaluated. A biobleaching experiment was carried out to compare the chlorine saving with pulp treated and untreated by the enzyme. Two-stage bleaching was employed, using a ClO2 chlorination and NaOH extraction (DE sequence). With the enzymatic treatment, in order to obtain the same value of Kappa number and brightness, respectively 28.5 and 30% less ClO2 was required in comparison to the enzymatically untreated samples.

Evaluation of Bond Strength of Self-adhesive Cements to Dentin With or Without Application of Adhesive Systems

Purpose: To evaluate the bond strength of indirect restorations to dentin using self-adhesive cements with and without the application of adhesive systems.Material and Methods: Seventy-two bovine incisors were used, in which the buccal surfaces were ground down to expose an area of dentin measuring a minimum of 4 x 4 mm. The indirect resin composite Resilab was used to make 72 blocks, which were cemented onto the dentin surface of the teeth and divided into 4 groups (n = 18): group 1: self-adhesive resin cement BiFix SE, applied according to manufacturer's recommendations; group 2: self-adhesive resin cement RelyX Unicem, used according to manufacturer's recommendations; group 3: etch-and-rinse Solobond M adhesive system + BiFix SE; group 4: etch-and-rinse Single Bond 2 adhesive system + RelyX Unicem. The specimens were sectioned into sticks and subjected to microtensile testing in a universal testing machine (EMIC DL-200MF). Data were subjected to one-way ANOVA and Tukey's test (alpha = 5%).Results: The mean values (+/- standard deviation) obtained for the groups were: group 1: 15.28 (+/- 8.17)(a), group 2: 14.60 (+/- 5.21)(a), group 3: 39.20 (+/- 9.98)(c), group 4: 27.59 (+/- 6.57)(b). Different letters indicate significant differences (ANOVA; p = 0.0000).Conclusion: The application of adhesive systems before self-adhesive cements significantly increased the bond strength to dentin. In group 2, RelyX Unicem associated with the adhesive system Single Bond 2 showed significantly lower mean tensile bond strengths than group 3 (BiFix SE associated with the etch-and-rinse Solobond M adhesive system).