Ion channel-like activity of the antimicrobial peptide tritrpticin in planar lipid bilayers

The cationic peptide tritrpticin (VRRFPWWWPFLRR, Trp3) has a broad action spectrum, acting against Gram-positive and Gram-negative bacteria, as well as some fungi, while also displaying hemolytic activity. We have studied the behavior of Trp3 in planar lipid bilayers (or black lipid membrane-BLM) and were able to demonstrate its ion channel-like activity. Channel-like activity was observed in negatively charged azolectin BLM as a sudden appearance of discrete current fluctuations upon application of a constant voltage across the membrane. Trp3 formed large conductance channels (500-2000 pS) both at positive and negative potentials. in azolectin bilayers, the predominant ion-channel activity was characterized by very regular and discrete current steps (corresponding to openings) of uniform amplitude, which exhibited relatively long residence times (of the order of seconds). Occasionally, multiple conductance steps were observed, indicating the simultaneous presence of more than one open pore. in bilayers of zwitterionic diphytanoylphosphatidyl choline (DPhk) Trp3 also showed ion-channel activity, but in a much less frequent and less prominent way. Studies of ion selectivity indicated that Trp3 forms a cation-selective channel. These results should contribute to the understanding of the molecular interactions and mechanism of action of Trp3 in lipid bilayers and biological membranes. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.Univ São Paulo, Inst Chem, Dept Biochem, Struct Biol Lab, BR-05513970 São Paulo, BrazilUniv São Paulo, Inst Biomed Sci, Dept Physiol & Biophys, BR-05389970 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

Binding affinities and activation of Asp712Ala and Cys100Ser mutated kinin B1 receptor forms suggest a bimodal scheme for the molecule of bound-DABK

AbstractMutant forms of kinin B1 receptor (B1R) and analogs of the full agonist des-Arg9-bradykinin (DABK) were investigated aiming to verify the importance of selected receptor residues and of each agonist-peptide residue in the specific binding and activation. Linked by a specific disulfide bond (Cys100–Cys650), the N-terminal (Nt) and the EC3 loop C-terminal (Ct) segments of angiotensin II (AngII) receptor 1 (AT1R) have been identified to form an extracellular site for binding the agonist Nt segment (Asp1 and Arg2 residues). Asp712 residue at the receptor EC3 loop binds the peptide Arg2 residue. By homology, a similar site might be considered for DABK binding to B1R since this receptor contains the same structural elements for composing the site in AT1R, namely the disulfide bond and the EC3 loop Asp712 residue. DABK, Alan-DABK analogs (n=Ala1-, Ala2-, Ala3-, Ala4-, Ala5-, Ala6-, Ala7-, Ala8-DABK), and other analogs were selected to binding wild-type, Asp712Ala and Cys100Ser mutated B1R receptors. The results obtained suggested that the same bimodal scheme adopted for AngII-AT1R system may be applied to DABK binding to B1R. The most crucial similarity in the two cases is that the Nt segments of peptides equally bind to the homologous Asp712 residue of both AT1R and B1R extracellular sites. Confirming this preliminary supposition, mutation of residues located at the B1R extracellular site as EC3 loop Asp712 and Cys100 caused the same modifications in biological assays observed in AT1R submitted to homologous mutations, such as significant weakening of agonist binding and reduction of post-receptor-activation processes. These findings provided enough support for defining a site that determines the specific binding of DABK to B1R receptors

Effect of polysaccharide capsule of the microalgae Staurastrum iversenii var. americanum on diffusion of charged and uncharged molecules, using EPR technique

The existence of a mucilaginous envelope, sheath or capsule is usual in many desmids, but few data concerning its function are available. Previous studies of the transport function and permeation of molecules through the algae capsules were done using the algae Spondylosium panduriforme and Nephrocytium lunatum, the Electron Paramagnetic Resonance (EPR) technique, and different spin labels. The results suggested that the capsule functions as a selective diffusion medium. In the present work charged and uncharged molecules (spin labels group A) and Staurastrum iversenii var. americanum (Desmids),whose alga presents a great mucilaginous capsule, were used. Charged nitroxide molecules similar to amino acids (spin labels group B) were also used allowing a better understanding of the electrostatic effect in the permeation process across the capsule. The role of the cell capsule in the solute diffusion was evaluated by determining the capsulated and decapsulated cell permeation times. The permeation times for all spin labels tested in the cells lacking capsules were always shorter than those containing this physical barrier. The decay times of spin labels group A observed for S. iversenii were compared to other studied algae. The results regarding the diffusion of charged spin labels group B suggested that the interaction of cell capsule occurs more strongly with negatively charged molecules than with positively charged ones. The results obtained in this work with spin labels group A confirm that the capsule is an essential structure for the cell, and that due to the polar interactions with the spin labels, it plays an important role in the selection of small molecules. Several parameters, mainly those of electrostatic nature, seem to control the permeation across the algal capsules of spin labels group B, showing that structures which are similar to amino acids could diffuse across the interior of the algal cell.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)USP Instituto de Física de São Carlos Departamento de Física e InformáticaUniversidade Federal de São Carlos Departamento de BotânicaUniversidade Federal de São Paulo (UNIFESP) Departamento de BiofísicaUNIFESP, Depto. de BiofísicaSciEL

First synthesis of a fully active spin-labeled peptide hormone

For the first time in the electron spin resonance (ESR) and peptide synthesis fields, a fully active spin-labeled peptide hormone was reported. the ESR spectra of this alpha-melanocyte stimulating hormone (alpha-MSH) analogue (acetyl-Toac(0)-alpha-MSH) where Toac is the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, suggested a pH-independent conformation and a more restricted movement comparatively to the free Toac, Owing to its equivalent biological potency in a skin pigmentation assay as compared to the native alpha-MSH and its unique characteristic (paramagnetic, naturally fluorescent and fully active), this analogue is of great potential for investigation of relevant physiological roles reported for a-MSH, (C) 1999 Federation of European Biochemical Societies.Universidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniv São Paulo, Inst Fis, BR-01498 São Paulo, BrazilUniv São Paulo, Inst Biociencias, Dept Fisiol, BR-01498 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

Application of Electron Paramagnetic Resonance Spectroscopy for Validation of the Novel (AN+DN) Solvent Polarity Scale

Based on solvation studies of polymers, the sum (1:1) of the electron acceptor (AN) and electron donor (DN) values of solvents has been proposed as an alternative polarity scale. To test this, the electron paramagnetic resonance isotropic hyperfine splitting constant, a parameter known to be dependent on the polarity/proticity of the medium, was correlated with the (AN+DN) term using three paramagnetic probes. The linear regression coefficient calculated for 15 different solvents was approximately 0.9, quite similar to those of other well-known polarity parameters, attesting to the validity of the (AN+DN) term as a novel “two-parameter” solvent polarity scale

Comparative Bioavailability Of Two Quetiapine Formulations In Healthy Volunteers After A Single Dose Administration

The study was performed to compare the bioavailability of two quetiapine 25 mg tablet formulations: the test formulation was quetiapine fumarate (kitapen®) manufactured by Cobalt Pharmaceuticals, Canada/ Arrow Farmacêutica Ltda* (Erowlabs). Seroquel® (quetiapine) from Astrazeneca Brazil was used as reference formulation. The study was conducted open with randomized two period crossover design and one week wash out period in 64 volunteers of both sexes. Plasma samples were obtained over a 48 hour interval. Quetiapine was analyzed by LC-MS-MS in the presence of quetiapine-D8 as internal standard. Plasma samples were obtained over a 48 hour interval. Quetiapine was analyzed by LC-MS-MS in the presence of quetiapine-D8 as internal standard. The mean ratio of parameters Cmax and AUC 0-t and 90% confidence intervals of correspondents were calculated to determine the bioequivalence. The means AUC 0-t for test and reference formulation were 432.41 ng.h/mL and 412.20 ng.h/mL, for AUC 0-∞ were 440.06 ng.h/mL and 418.90 ng.h/mL and, for Cmax 126.94 ng/mL and 108.71 ng/mL, respectively. Geometric mean of quetiapine (kitapen®)/Seroquel® 25 mg individual percent ratio was 97.68% AUC 0-t, 97.47% for AUC 0-∞ and 90.68% for C max. The 90% confidence intervals were 92.67 - 102.96%, 92.53 - 102.67%, 83.37 - 98.64%, respectively. Since the 90% confidence intervals for C max, AUC 0-t and AUC 0-∞ were within the 80 - 125% interval proposed by Food and Drug Administration, it was concluded that quetiapine (kitapen®) 25 mg tablet was bioequivalent to Seroquel® 25 mg tablet according to both the rate and extent of absorption. © 2011 Junior EA, et al.38178181Barrett, B., Capek, H.M., Huclova, J., Borek-Dohalsky, V., Fejt, P., Validated HPLC-MS/MS method for determination of quetiapine in human plasma (2007) Journal of Pharmaceutical and Biomedical Analysis, 44, pp. 498-505DeVane, C.L., Nemeroff, C.B., Clinical Pharmacokinetics of quetiapine: An Atypical Antipsychotic (2001) Clinical Pharmacokinet, 40, pp. 509-522Kasper, S., Müller-Spahn, F., Review of quetiapine and its clinical applications in schizophrenia (2000) Expert Opin Pharmacother, 1, pp. 783-801Tilden, D., Aristides, M., Meddis, D., Burns, T., An economic assessment of quetiapine and haloperidol in patients with schizophrenia only partially responsive to conventional antipsychotics (2002) Clin Ther., 24, pp. 1648-1667Mario, A., Michael, E., The Role of Quetiapine Extended Release in the Treatment of Bipolar Depression (2010) Adv Ther, 27, pp. 1-11Keck, P., McIntyre, R., Shelton, R., Bipolar depression: Best practices for the outpatient (2007) CNS Spectr., 12, pp. 1-16Judd, L., Akishal, H., Schettler, P., The long-term natural history of the weekly symptomatic status of bipolar I disorder (2002) Arch Gen Psychiatry., 59, pp. 530-537Goldstein, J.M., Atypical antipsychotic drugs: Beyond acute psychosis, new directions (1999) Emerging Drugs, 4, pp. 127-151Abi-Dargham, A., Laruelle, M., Aghajanian, G.K., Charney, D., Krystal, J., The role of serotonin in the pathophysiology and treatment of schizophrenia (1997) J Neuropsychiatry Clin Neurosci, 9, pp. 1-17Kapur, S., Remington, G., Serotonin-dopamine interaction and its relevance to schizophrenia (1996) Am J Psychiatry, 153, pp. 466-476Calabrese, J.R., Keck Jr., P.E., McFadden, W., Minkwitz, M., Ketter, T.A., Weisler, R.H., Cutler, A.J., Mullen, J., A randomized, doubleblind, placebo-controlled trial of quetiapine in the treatment of bipolar I or II depression (2005) Am J Psychiatry, 162, pp. 1351-1360Copolov, D.L., Kowalcyk, B., A multicentre, double-blind, randomized comparison of quetiapine and haloperidol in schizophrenia (2000) Psychol Med, 30, pp. 95-105Figueroa, C., Brecher, M., Hamer-Maansson, J., Pharmacokinetic profiles of extended release quetiapine fumarate compared with quetiapine immediate release (2009) Prog Neuropsychopharmacol Biol Psychiatry, 33, pp. 199-204Goldstein, J.M., Litwin, L.C., Sutton, E.B., Malick, J.B., Seroquel: Electrophysiological profile of a potential atypical antipsychotic (1993) Psychopharmacology, 112, pp. 293-298Kasper, S., Tauscher, J., Küfferle, B., Barnas, C., Pezawas, L., Dopamine and serotonin-receptors in schizophrenia: Results of imaging-studies and implications for pharmacology in schizophrenia (1999) Eur. Arch. Psychiatry Clin. Neurosci., 249, pp. 83-89Peuskens, J., A comparison of quetiapine and chlorpromazine in the treatment of schizophrenia (1997) Acta Psychiatr Scand, 96, pp. 265-273Saller, F., Salama, A.I., Seroquel: Biochemical profile of a potential atypical antipsychotic (1993) Psychopharmacology, 112, pp. 285-292Thase, M.E., McFadden, W., Weisler, R., Efficacy of quetiapine monotherapy in bipolar I and II depression: A double-blind, placebo-controlled study (2006) J Clin Psychopharmacol, 26, pp. 600-609Vieta, E., Mullen, J., Brecher, M., Paulsson, B., Jones, M., Quetiapine monotherapy for mania associated with bipolar disorder: Combined analysis of two international, double-blind, randomised, placebo-controlled studies (2005) Curr Med Res Opin, 21, pp. 923-93

Importance of the solvation degree of peptide-resin beads for amine groups determination by the picric acid method

The classic and important picric acid method used in polymers biochemical and chemical fields of polymers for amine group quantification was chosen in this work as a model for evaluating the influence of the resin bead solvation during an analytical procedure. It was observed that this method, proposed almost three decades ago, failed to quantify amine groups of peptidyl-resin containing aggregating and polar sequence. This was due to inefficient solvation of resin beads when only CH2Cl2 was used for picrate anion binding and subsequent washing steps. It was demonstrated that the use of CH2Cl2/DMF (dimethylformamide) and CH2Cl2/EtOH solutions during these steps allows correct determination of peptidyl-resin amine groups. Besides the importance for the solid phase peptide synthesis methodology itself, these findings also represent the first quantitative demonstration of the relationship between solvation degree and the efficiency of a polymer-supported analytical method.O clássico e importante método do ácido pícrico, usado nas áreas de bioquímica e química de polímeros para a quantificação de grupos aminos, foi escolhido neste trabalho como modelo para investigar a importância do grau de solvatação de grãos poliméricos durante o protocolo analítico. Verificou-se que este método, proposto há cerca de três décadas atrás, falha na quantificação de grupos amino de peptidil-resinas contendo seqüência agregante e polar. Isto ocorre devido à solvatação insuficiente dos grãos quando apenas o CH2Cl2 é utilizado na etapa de ligação do ânion picrato e na subsequente etapa de lavagem. Demonstrou-se que a utilização nestas etapas, de soluções de CH2Cl2/DMF (dimetilformamida) e de CH2Cl2/EtOH permite uma determinação correta dos grupos amínicos de peptídil-resinas. Além da importância em si para o método da síntese de peptídeos em fase sólida, estes resultados representam também a primeira comprovação experimental da correlação quantitativa existente entre o grau de solvatação e a eficiência de um método analítico efetuado em grãos de resinas.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo (UNIFESP) Departamento de BiofísicaUNIFESP, Depto. de BiofísicaSciEL

Use of FTIR in the obtention of resins and peptides synthesis in solid phase

Despite the increase in peptide chain aggregation, which decreases the rate of coupling reactions, the synthesis and use of very highly substituted resins still remains as a controversial point in the SPPS, due to its clear economical advantages (lesser solvent consumption and higher amount of peptide per synthesis). In order to better investigate the synthesis and the use of very highly substituted resins, the FTIR, NMR and EPR were compared. By FTIR techniques it was possible to follow all the steps of resin synthesis and the factors affecting the aggregation of the chains inside the peptidil-BHAR and MBHAR.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Estadual Paulista Instituto de Química Departamento de Bioquímica e Tecnologia QuímicaUniversidade Federal de São Paulo (UNIFESP) Departamento de BiofísicaUNIFESP, Depto. de BiofísicaSciEL

Use of the same polymer for synthesis and purification of peptides

This work reveals an uncommon but valuable biotechnological approach regarding the use of a same polymer (benzhydrylamine-resin, BHAR) for synthesis and anion exchange purification of peptides. Initially, the octapeptide DRVYIHPF-NH2 was synthesized in 1% and 3% cross-linked BHAR, attaching 2.5 mmol g-1 ammonium groups. Due certainly to its less rigid polymeric backbone, higher synthesis yield (about 80%) was achieved with the former resin. Next, the negatively charged peptides DEVYEHPF-NH2 and DEVYEDPF-NH2 (-1 and -3 in neutral pH, respectively), both synthesized in 1% BHAR were submitted to chromatographic separation test in this same type of resin (1% and 3%). Following comparative results of peptide synthesis and swelling data of resin beads obtained by microscopy, an improved separation of both peptides occurred with 1% BHAR batch. These findings demonstrated that BHAR applied so far for peptide synthesis, when containing high amount of positively charged ammonium groups, can be also used alternatively as a solid support for chromatographic purification of this type of biological molecule.Este trabalho revela uma estratégia biotecnológica incomum mas valiosa e relacionada com o uso de um mesmo polímero (benzidrilamino-resina, BHAR) para fins de síntese e purificação por troca aniônica de peptídeos. Inicialmente, o octapeptídeo DRVYIHPF-NH2 foi sintetizado em BHAR com 1% e 3% de intercruzamento e contendo 2,5 mmol g-1 de grupos amônio. Devido certamente a uma menor rigidez da sua estrutura polimérica, um maior rendimento na síntese (cerca de 80%) foi obtido com a primeira resina. A seguir, os peptídeos DEVYEHPF-NH2 e DEVYEDPF-NH2 carregados negativamente (-1 e -3 em pH neutro, respectivamente), ambos sintetizados em 1% BHAR, foram submetidos a um teste de separação cromatográfica no mesmo tipo de resina (1% e 3%). Concordante com resultados da síntese de peptídeos e de valores de inchamento dos grãos das resinas, obtidos por microscopia, uma melhor separação entre ambos os peptídeos ocorreu com o lote de 1% BHAR. Estes achados demonstraram que a BHAR, aplicada até o momento somente para a síntese peptídica, se contiver elevado teor de grupos amônio positivos, pode ser utilizada alternativamente como suporte sólido para purificação cromatográfica deste tipo de molécula biológica.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo (UNIFESP) Departmento de BiofísicaUNIFESP, Departmento de BiofísicaSciEL