A proposed EPR approach to evaluating agonist binding site of a peptide receptor

Angiotensin II (Ang II) and its transmembrane AT(1) receptor were selected in order to test an innovative strategy that might allow the assessment of the agonist binding site in the receptor molecule. With the use of the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) paramagnetic probe, a biologically active agonist (TOAC(1)-Ang II), as well as an inactive control (TOAC(4)-Ang II) analogs were mixed in solution with various synthesized AT(1) fragments. Comparative intermolecular interactions, as estimated by analyzing the EPR spectra of solutions, suggested the existence of an agonist binding site containing a sequence composed of portions of the N-terminal (13-17) and the third extracellular loop (266-278) fragments of the AT(1) molecule. Therefore, this combined EPR-TOAC approach shows promise as an alternative for use also in other applications related to specific intermolecular association processes.

Detection and Characterization of Cholesterol-Oxidized Products Using HPLC Coupled to Dopant Assisted Atmospheric Pressure Photoionization Tandem Mass Spectrometry

Oxidation of cholesterol (Ch) by a variety of reactive oxygen species gives rise mainly to hydroperoxides and aldehydes. Despite the growing interest in Ch-oxidized products, the detection and characterization of these products is still a matter of concern. In this work, the main Ch-oxidized products, namely, 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide (7 alpha-OOH), 3 beta-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 alpha-hydroperoxide (6 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH), and 3 beta-hydroxy-5 beta-hydroxy-B-norcholestane-6 beta-carboxaldehyde (ChAld), were detected in the same analysis using high-performance liquid chromatography (HPLC) coupled to dopant assisted atmospheric pressure photoionization tandem mass spectrometry. The use of selected reaction monitoring mode (SRM) allowed a sensitive detection of each oxidized product, while the enhanced product ion mode (EPI) helped to improve the confidence of the analyses. Isotopic labeling experiments enabled one to elucidate mechanistic features during fragmentation processes. The characteristic fragmentation pattern of Ch-oxidized products is the consecutive loss of 1120 molecules, yielding cationic fragments at m/z 401, 383, and 365. Homolytic scissions of the peroxide bond are also seen. With (18)O-labeling approach, it was possible to establish a fragmentation order for each isomer. The SRM transitions ratio along with EPI and (18)O-labeled experiments give detailed information about differences for water elimination, allowing a proper discrimination between the isomers:Phis is of special interest considering the emerging role of Ch-oxidized products in the development of diseases.

Microwave-assisted solid-phase peptide synthesis at 60 degrees C: alternative conditions with low enantiomerization

Several conditions have been used in the coupling reaction of stepwise SPPS at elevated temperature (SPPS-ET), but we have elected the following as our first choice: 2.5-fold molar excess of 0.04-0.08 M Boc or Fmoc-amino acid derivative, equimolar amount of DIC/HOBt (1:1)or TBTU/DIPEA(1:3), 25% DMSO/toluene, 60 degrees C, conventional heating. In this study, aimed to further examine enantiomerization under such condition and study the applicability of our protocols to microwave-SPPS, peptides containing L-Ser, L-His, L-Cys and/or L-Met were manually synthesized traditionally, at 60 degrees C using conventional heating and at 60 degrees C using microwave heating. Detailed assessment of all crude peptides (in their intact and/or fully hydrolyzed forms) revealed that, except for the microwave-assisted coupling of L-Cys, all other reactions occurred with low levels of amino acid enantiomerization (<2%). Therefore, herein we (i) provide new evidences that our protocols for SPPS at 60 degrees C using conventional heating are suitable for routine use, (ii) demonstrate their appropriateness for microwave-assisted SPPS by Boc and Fmoc chemistries, (iii) disclose advantages and limitations of the three synthetic approaches employed. Thus, this study complements our past research on SPPS-ET and suggests alternative conditions for microwave-assisted SPPS. Copyright (C) 2009 European Peptide Society and John Wiley & Sons, Ltd.

Effect of unsaturated fatty acids on myocardial performance, metabolism and morphology

Diets rich in saturated fatty acids are one of the most important causes of atherosclerosis in men, and have been replaced with diets rich in unsaturated fatty acids (UFA) for the prevention of this disorder. However, the effect of UFA on myocardial performance, metabolism and morphology has not been completely characterized. The objective of the present investigation was to evaluate the effects of a UFA-rich diet on cardiac muscle function, oxidative stress, and morphology. Sixty-day-old male Wistar rats were fed a control (N = 8) or a UFA-rich diet (N = 8) for 60 days. Myocardial performance was studied in isolated papillary muscle by isometric and isotonic contractions under basal conditions after calcium chloride (5.2 mM) and ss-adrenergic stimulation with 1.0 mu M isoproterenol. Fragments of the left ventricle free wall were used to study oxidative stress and were analyzed by light microscopy, and the myocardial ultrastructure was examined in left ventricle papillary muscle. After 60 days the UFA-rich diet did not change myocardial function. However, it caused high lipid hydroperoxide (176 +/- 5 vs 158 +/- 5, P < 0.0005) and low catalase (7 +/- 1 vs 9 +/- 1, P < 0.005) and superoxide-dismutase (18 +/- 2 vs 27 +/- 5, P < 0.005) levels, and discrete morphological changes in UFA-rich diet hearts such as lipid deposits and mitochondrial membrane alterations compared to control rats. These data show that a UFA-rich diet caused myocardial oxidative stress and mild structural alterations, but did not change mechanical function.

Cloning, sequencing and expression analysis of the equine hepcidin gene by real-time PCR

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

Influence of experimental inflammatory response on hepatic hepcidin gene expression and plasma iron concentration in sheep

Hepcidin is a highly conserved disulfide-bonded peptide that plays a central role in iron homeostasis. During systemic inflammation, hepcidin up-regulation is responsible for hypoferremia. This study aimed to analyze the influence of the inflammatory process induced by complete Freund's adjuvant (CFA) or lipopolysaccharide (LPS) on the liver expression of hepcidin mRNA transcripts and plasma iron concentration of sheep. The expression levels of hepcidin transcripts were up-regulated after CFA or LPS. Hypoferremic response was observed at 12 h (15.46 +/- 6.05 mu mol/L) or 6 h (14.59 +/- 4.38 mu mol/L) and iron reached its lowest level at 96 h (3.08 +/- 1.18 mu mol/L) or 16 h (4.06 +/- 1.58 mu mol/L) after CFA administration or LPS infusion, respectively. This study demonstrated that the iron regulatory hormone hepcidin was up-regulated in sheep liver in response to systemic inflammation. These findings extend our knowledge on the relationship between the systemic inflammatory response, hepcidin and iron, and provide a starting point for additional studies on iron metabolism and the inflammatory process in sheep. (C) 2011 Elsevier B.V. All rights reserved.

Probing the Specificity of Binding to the Major Nuclear Localization Sequence-binding Site of Importin-alpha Using Oriented Peptide Library Screening

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

Structural and functional characterization of two novel peptide toxins isolated from the venom of the social wasp Polybia paulista

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

Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae

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

Solid-phase peptide synthesis in highly loaded conditions

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

Correlation between solvation of peptide-resins and solvent properties

The solvation properties of model resin and peptide-resins measured in ca. 30 solvent systems correlated better with the sum of solvent electron acceptor (AN) and electron donor (DN) numbers, in 1:1 proportion, than with other solvent polarity parameters. The high sensitivity of the (AN+DN) term to detect differentiated solvation behaviors of peptide-resins, taken as model of heterogeneous and complex solutes, seems to be in agreement with the previously proposed two-parameter model, where the sum of the Lewis acidity and Lewis basicity characters of solvent are proposed for scaling solvent effect. Besides these physicochemical aspects regarding solute-solvent interactions, important implications of this study for the solid phase peptide synthesis were also observed. Each class of peptide-resin displayed a specific salvation profile that was dependent on the amount and the nature of the resin-bound peptide sequence. Plots of resin swelling versus solvent (AN+DN) values allowed the visualization of a maximum salvation region characteristic for each class of resin. This strategy facilitates the selection of solvent systems for optimal solvation conditions of peptide chains in every step of the entire synthesis cycle. Moreover, only the AN and DN concepts allow the understanding of rules for solvation/shrinking of peptide-resins when in homogeneous or in heterogeneous mixed solvents.

Metabolism of stevioside by chickens

In intubation experiments (643-1168 mg per animal), most of the stevioside administered to chickens was recovered unchanged in the excreta, and only about 2% was converted into steviol. Neither stevioside nor steviol could be found in the blood. In chronic studies (667 mg of stevioside/kg of feed) with laying hens and meat-type chickens, no significant differences were found in feed uptake, weight gain, and feed conversion as the result of stevioside administration. The egg production and egg composition of laying hens were not influenced. Most of the stevioside taken up was found untransformed in the excreta, and about 21.5% or 7.3% was converted to steviol by meat-type chickens or laying hens, respectively. No stevioside or steviol could be detected in the blood or in the eggs of the different groups of animals. In anaerobic incubation experiments with chicken excreta, only a 20% conversion of stevioside into steviol was found. No harmful effects were observed in the chronic stevioside supplementation experiments nor in the intubation experiments in which very high stevioside doses were given.

Correlation between the mobility of spin-labeled peptide chains and resin solvation: An approach to optimize the synthesis of aggregating sequences

Resin solvation properties affect the efficiency of the coupling reactions in solid-phase peptide synthesis. Here we report a novel approach to evaluate resin solvation properties, making use of spin label electron paramagnetic resonance (EPR) spectroscopy. The aggregating VVLGAAIV and ING sequences were assembled in benzhydrylamine-resin with different amino group contents (up to 2.6 mmol/g) to examine the extent of chain association within the beads. These model peptidyl-resins were first labeled at their N-terminus with the amino acid spin label 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac). Their solvation properties in different solvents were estimated, either by bead swelling measurement or by assessing the dynamics of their polymeric matrixes through the analysis of Toac EPR spectra, and were correlated with the yield of the acylation reaction. In most cases the coupling rate was found to depend on bead swelling. Comparatively, the EPR approach was more effective. Line shape analysis allowed the detection of more than one peptide chain population, which influenced the reaction. The results demonstrated the unique potential of EPR spectroscopy not only for improving the yield of peptide synthesis, even in challenging conditions, but also for other relevant polymer-supported methodologies in chemistry and biology.

EPR investigation of the influence of side chain protecting groups on peptide-resin solvation of the Asx and Glx model containing peptides

In spite of all progressive efforts aiming to optimize SPPS, serious problems mainly affecting the assembly of aggregating sequences have persisted. Following the study intended to unravel the complex solvation phenomenon of peptide-resin beads, the XING and XAAAA model aggregating segments were labeled with a paramagnetic probe and studied via EPR spectroscopy. Low and high substituted resins were also comparatively used, with the X residue being Asx or Glx containing the main protecting groups used in the SPPS. Notably, the cyclo-hexyl group used for Asp and Glu residues in Boc-chemistry induced greater chain immobilization than its tert-butyl partner-protecting group of the Fmoc strategy. Otherwise, the most impressive peptide chain immobilization occurred when the large trytil group was used for Asn and Gln protection in Fmoc-chemistry. These surprising results thus seem to stress the possibility of the relevant influence of the amino-acid side chain protecting groups in the overall peptide synthesis yield. (C) 2007 Elsevier Ltd. All rights reserved.

EFFECT OF EXERCISE DURING PREGNANCY AND DAM AGE ON MATERNAL BLOOD-CHEMISTRY AND FETAL GROWTH

In order to determine the effect of maternal exercise on maternal nutritional status and fetal growth, young (Y = 45-50 days old) Wistar rats were divided into 4 groups of 5 to 8 animals: control pregnant (CP), control non-pregnant (CNP), exercise-trained (swimming 1 h/day, 5 days/week, for 19 days) pregnant (TP) and exercise-trained non-pregnant (TNP). Four equivalent groups of adult rats (A - 90-100 days old) were also formed. Serum glucose, total protein, albumin, hematocrit and liver glycogen were determined in female rats and pups. There were no statistical differences in serum glucose, total protein and albumin levels, litter size ot birth weight among exercise-trained animals, controls and their respective pups. Hematocrit was significantly lower in pups of exercise-trained young rats than in all other groups (YCP = 38.6 +/- 3.0; YTP = 32.6 +/- 2.1; ACP = 39.0 +/- 2.5; ATP = 39.2 +/- 2.9%). Liver glycogen levels were lower in pregnant than in non-pregnant rats but similar in exercise-trained and control rats of the same age and physiological status (YCNP = 4.1 +/- 0.2; YCP = 2.7 +/- 0.9; YTNP = 4.9 +/- 0.8; YTP = 2.7 +/-0.4; ACNP = 6.1 +/- 0.6; ACP = 3.1 +/- 0.8; ATNP = 6.6 +/- 0.8; ATP = 2.2 +/- 0.9 mg/100 mg). We conclude that pups of adult female rats are spared from the effects of this kind of exercise training during pregnancy. on the other hand, it appears that maternal adaptations to exercise training in young rats are able to preserve only some aspects of pup metabolism.