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The (1-42) β-amyloid peptide is a main component of the plaques found in the brain of patients suffering from the Alzheimer's disease. As the single substitution of Glu for Gln at position 22 of this peptide seems to be responsible for the manifestation of the more severe amyloidosis (Dutch-type), we decided to evaluate the aggregation characteristics of peptide analogs interchanging Glu and Gln residues at positions 22 and also 15 in the minor (12-24) (VHHQ(15)KLVFFAE(22)DV) fragment. The Q15Q22, E15E22, E15Q22 and the native Q15E22 were compared to the (1-42) β-amyloid peptide in terms of fibril or structured aggregates formation propensity. In contrast to a rather similar solubility data measured of all analogs, fluorescence and light scattering methods indicated that only Q15E22 and Q15Q22 displayed relevant fibril formation capacity. Conversely, E15E22 and E15Q22 were not capable of the formation of this type of structure thus suggesting a key role for the Q(15) residue in the unique aggregation characteristic of the β-amyloid peptide. [ABSTRACT FROM AUTHOR]

We investigate the physicochemical effects of pyroglutamination on the QHALTSV-NH 2 peptide, a segment of cytosolic helix 8 of the human C-X-C chemokine G-protein-coupled receptor type 4 (CXCR4). This modification, resulting from the spontaneous conversion of glutamine to pyroglutamic acid, has significant impacts on the physicochemical features of peptides. Using a static approach, we compared the transformation in different conditions and experimentally found that the rate of product formation increases with temperature, underscoring the need for caution during laboratory experiments to prevent glutamine cyclization. Circular dichroism experiments revealed that the QHALTSV-NH 2 segment plays a minor role in the structuration of H8 CXCR4; however, its pyroglutaminated analogue interacts differently with its chemical environment, showing increased susceptibility to solvent variations compared to the native form. The pyroglutaminated analogue exhibits altered behavior when interacting with lipid models, suggesting a significant impact on its interaction with cell membranes. A unique combination of atomic force microscopy and infrared nanospectroscopy revealed that pyroglutamination affects supramolecular self-assembly, leading to highly packed molecular arrangements and a crystalline structure. Moreover, the presence of pyroglumatic acid has been found to favor the formation of amyloidogenic aggregates. Our findings emphasize the importance of considering pyroglutamination in peptide synthesis and proteomics and its potential significance in amyloidosis.

Objectives: Evaluate molecularly the role of P 11 -4 self-assembly peptide in dentin remineralization and its interaction with collagen I., Methods: The calcium-responsive P 11 -4 peptide was analyzed by intrinsic fluorescence emission spectrum, circular dichroism spectrum (CD), and atomic force microscope (AFM). Differential light scattering was used to monitor the nucleation growth rate of calcium phosphate nanocrystals in the absence or in the presence of P 11 -4. AFM was used to analyze the radial size (nm) of calcium phosphate nanocrystals formed in the absence or in the presence of P 11 -4, as well as to verify the spatial structure of P 11 -4 in the absence or in the presence of Ca 2+ ., Results: The interaction of Ca 2+ with the P 11 -4 (K D = 0.58 ± 0.06 mM) promotes the formation of β-sheet antiparallel structure, leads to its precipitation in saturated solutions of Ca/P = 1.67 and induces the formation of parallel large fibrils (0.6 - 1.5 µm). P 11 -4 organized the HAP nucleation by reducing both the growth rate and size variability of nanocrystals, analyzed by the F test (p < 0.0001, N = 30). P 11 -4 interacts (K D = 0.75 ± 0.06 μM) with the KGHRGFSGL motif present at the C-terminal collagen telopeptide domain. P 11 -4 also increased the amount of HAP and collagen in the MDPC-23 cells., Significance: The presented data propose a mechanism that will help future clinical and/or basic research to better understand a molecule able to inhibit structural collagen loss and help the impaired tissue to remineralize., (Copyright © 2023 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

Bradykinin (BK) is a peptide hormone that plays a crucial role in blood pressure control, regulates inflammation in the human body, and has recently been implicated in the pathophysiology of COVID-19. In this study, we report a strategy for fabricating highly ordered 1D nanostructures of BK using DNA fragments as a template for self-assembly. We have combined synchrotron small-angle X-ray scattering and high-resolution microscopy to provide insights into the nanoscale structure of BK-DNA complexes, unveiling the formation of ordered nanofibrils. Fluorescence assays hint that BK is more efficient at displacing minor-groove binders in comparison with base-intercalant dyes, thus, suggesting that interaction with DNA strands is mediated by electrostatic attraction between cationic groups at BK and the high negative electron density of minor-grooves. Our data also revealed an intriguing finding that BK-DNA complexes can induce a limited uptake of nucleotides by HEK-293t cells, which is a feature that has not been previously reported for BK. Moreover, we observed that the complexes retained the native bioactivity of BK, including the ability to modulate Ca 2+ response into endothelial HUVEC cells. Overall, the findings presented here demonstrate a promising strategy for the fabrication of fibrillar structures of BK using DNA as a template, which keep bioactivity features of the native peptide and may have implications in the development of nanotherapeutics for hypertension and related disorders.

The renin-angiotensin system (RAS) is a key regulator of human arterial pressure. Several of its effects are modulated by angiotensin II, an octapeptide originating from the action of angiotensin-I converting enzyme (ACE) on the decapeptide angiotensin-I. ACE possess two active sites (nACE and cACE) that have their own kinetic and substrate specificities. ACE inhibitors are widely used as the first-line treatment for hypertension and other heart-related diseases, but because they inactivate both ACE domains, their use is associated with serious side effects. Thus, the search for domain-specific ACE inhibitors has been the focus of intense research. Angiotensin (1-7), a peptide that also belongs to the RAS, acts as a substrate of nACE and an inhibitor of cACE. We have synthetized 15 derivatives of Ang (1-7), sequentially removing the N-terminal amino acids and modifying peptides extremities, to find molecules with improved selectivity and inhibition properties. Ac-Ang (2-7)-NH 2 is a good ACE inhibitor, resistant to cleavage and with improved cACE selectivity. Molecular dynamics simulations provided a model for this peptide's selectivity, due to Val 3 and Tyr 4 interactions with ACE subsites. Val 3 has an important interaction with the S 3 subsite, since its removal greatly reduced peptide-enzyme interactions. Taken together, our findings support ongoing studies using insights from the binding of Ac-Ang (2-7)-NH 2 to develop effective cACE inhibitors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Peptide-based hydrogels have attracted much attention due to their extraordinary applications in biomedicine and offer an excellent mimic for the 3D microenvironment of the extracellular matrix. These hydrated matrices comprise fibrous networks held together by a delicate balance of intermolecular forces. Here, we investigate the hydrogelation behavior of a designed decapeptide containing a tetraleucine self-assembling backbone and fibronectin-related tripeptides near both ends of the strand. We have observed that this synthetic peptide can produce hydrogel matrices entrapping >99% wt/vol % water. Ultrastructural analyses combining atomic force microscopy, small-angle neutron scattering, and X-ray diffraction revealed that amyloid-like fibrils form cross-linked networks endowed with remarkable thermal stability, the structure of which is not disrupted up to temperatures >80 °C. We also examined the interaction of peptide hydrogels with either NIH3T3 mouse fibroblasts or HeLa cells and discovered that the matrices sustain cell viability and induce morphogenesis into grape-like cell spheroids. The results presented here show that this decapeptide is a remarkable building block to prepare highly stable scaffolds simultaneously endowed with high water retention capacity and the ability to instruct cell growth into tumor-like spheroids even in noncarcinoma lineages.

Although the existence of the renin-angiotensin system (RAS) in the bone marrow is clear, the exact role of this system in hematopoiesis has not yet been fully characterized. Here the direct role of angiotensin II (AngII) in hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/monocyte progenitors (GMPs), and megakaryocytes/erythroid progenitors (MEPs), using a system of coculture with stromal S17 cells. Flow cytometry analysis showed that AngII increases the percentage of HSC and GMP, while reducing CMP with no effect on MEP. According to these data, AngII increased the total number of mature Gr-1 + /Mac-1 + cells without changes in Terr119 + cells. AngII does not induce cell death in the population of LSK cells. In these populations, treatment with AngII decreases the expression of Ki67 + protein with no changes in the Notch1 expression, suggesting a role for AngII on the quiescence of immature cells. In addition, exposure to AngII from murine bone marrow cells increased the number of CFU-GM and BFU-E in a clonogenic assay. In conclusion, our data showed that AngII is involved in the regulation of hematopoiesis with a special role in HSC, suggesting that AngII should be evaluated in coculture systems, especially in cases that require the expansion of these cells in vitro, still a significant challenge for therapeutic applications in humans., (© 2021 International Federation for Cell Biology.)

Objectives: This study evaluated the cell viability and expression of different major genes involved in mineralization in odontoblast-like cells exposed to sodium trimetaphosphate (STMP). It was also investigated the influence of STMP on the rate of calcium phosphate crystal growth, its anti-proteolytic action against the enzymatic degradation of type I collagen, the binding mechanism of STMP to collagen fibrils, and the potential mechanism to induce collagen stabilization., Methods: Immortalized rat odontoblast MDPC-23 cells were cultured. Cell viability was assessed by trypan blue staining, and the changes in gene expression balance induced by STMP were assessed by quantitative reverse transcription (qRT) PCR assays. Crystalline particle formation was monitored by light-scattering detectors to estimate pH variation and the radial size of the crystalline particles as a function of reaction time (pH 7.4, 25°C) in the presence of STMP in supersaturated calcium phosphate solution (Ca/P=1.67). Images were obtained under atomic force microscopy (AFM) to measure the particle size in the presence of STMP. A three-point bending test was used to obtain the elastic modulus of fully demineralized dentin beams after immersion in STMP solution. The binding mechanism of STMP to collagen fibrils and potential stabilization mechanism was assessed with circular dichroism spectrometry (CD). The data were analyzed statistically (α=0.05)., Results: STMP had no significant influence on the cell viability and gene expression of the MDPC-23 cells. STMP greatly increased the rate of crystal growth, significantly increasing the average radial crystal size. AFM corroborated the significant increase of STPM-treated crystal size. Mineralized collagen I fibrils exhibited less collagenase degradation with lower STMP concentration. CD analysis demonstrated changes in the conformational stability after STMP binding to type I collagen., Significance: The increased resistance of collagen against the proteolytic activity of collagenases appears to be related to the conformational change induced by STMP binding in collagen I and the STMP capacity for promoting biomimetic mineralization in type I collagen fibrils., (Copyright © 2020 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

Although antibodies are considered critical for malaria protection, little is known about the mechanisms/factors that maintain humoral immunity, especially regarding the induction and maintenance of memory B cells over time. In Brazilian endemic areas, this is the first time that the profile of antibody responses and the occurrence of antigen-specific memory B cells (MBC) against P vivax were investigated during acute malaria and up to six months after parasite clearance. For this, we selected two peptides, PvAMA-1 (S290-K307) and PvMSP-9 (E795-A808) , which represent the apical membrane antigen-1 and merozoite surface protein-9 of P vivax, respectively. Both peptides were previously described as containing linear B-cell epitopes. Our findings were as follows: 1-both peptides were recognized by IgG antibodies at a high frequency (between 24% and 81%) in all study groups; 2-in the absence of infection, the IgG levels remained stable throughout 6 months of follow-up; and 3-PvAMA-1 (S290-K307) and PvMSP-9 (E795-A808) -specific MBCs were detected in all individual groups in the absence of reinfection throughout the follow-up period, suggesting long-lived MBC. However, no positive association was observed between malaria-specific antibody levels and frequency of MBCs over time. Taken together, these results suggest that peptides can be, in the future, an alternative strategy to polypeptidic vaccine formulation., (© 2020 John Wiley & Sons Ltd.)

A strategy that has been gaining increased application for the study of the conformation, dynamics, orientation, and physicochemical properties of peptides is labeling with the paramagnetic amino acid TOAC. This approach was used to gain a deeper understanding on the mechanism of action of the antimicrobial peptide tritrpticin (TRP3). TRP3 was labeled with TOAC at the N-terminus (prior to V 1 , TOAC 0 -TRP3) or internally (replacing P 5 , TOAC 5 -TRP3). Functional studies showed that labeling led to peptides with higher activity against Gram-positive bacteria and lower hemolytic activity with respect to TRP3. Peptide-induced model membranes permeabilization and ion channel-like activity studies corroborated the functional assays qualitatively, showing higher activity of the peptides against negatively charged membranes, which had the purpose of mimicking bacterial membranes. TOAC presented a greater freedom of motion at the N-terminus than at the internal position, as evinced by EPR spectra. EPR and fluorescence spectra reported on the peptides conformational properties, showing acquisition of a more packed conformation in the presence of the secondary structure-inducing solvent, TFE. CD studies showed that TOAC 0 -TRP3 acquires a conformation similar to that of TRP3, both in aqueous solution and in TFE, while TOAC 5 -TRP3 presents a different conformation in all environments. While the mechanism of action of TRP3 was impacted to some extent by TOAC labeling at the N-terminus, it did change upon replacement of P 5 by TOAC. The results demonstrated that TOAC-labeling could be used to modulate TRP3 activity and mechanism of action and, more importantly, the critical role of P 5 for TRP3 pore formation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: Carboxypeptidase M (CPM) is a glycosylphosphatidylinositol anchored enzyme that plays an important role in the kallikrein-kinin system (KKS). CPM catalytic domain hydrolyzes Arg from C-terminal peptides (i.e., bradykinin and kallidin), generating des-Arg-kinins, the agonists of B 1 receptor (B 1 R). It is known that CPM and kinin B 1 R are co-localized in the plasma membrane microdomains, where they interact with each other, facilitating receptor signaling., Aims: We hypothesized here that this CPM-B 1 R interaction could also affect the activity of the enzyme., Methods: Thus, in this work, we evaluated the impact of B 1 R presence or absence on CPM activity and expression, using primary culture of microvascular endothelial cells from wild-type, kinin B 1 R knockout mice (B 1 -/- ), and transgenic rats overexpressing B 1 receptor exclusively in the endothelium. In addition, HEK293T cells, as wells as B 1 -/- primary culture of endothelial cells, both transfected with B 1 R, were also used., Results: CPM expression and activity were downregulated in cells of knockout mice compared to control and this reduction was rescued after B 1 R transfection. Cells overexpressing B 1 R presented higher levels of CPM mRNA, protein, and activity. This profile was reverted by pre-incubation with the B 1 R antagonist, R715, in highly expressing receptor cells., Conclusions: Our data show that kinin B 1 R positively modulates both CPM expression and activity, suggesting that CPM-B 1 R interaction in membrane microdomains might affect enzyme activity, beyond interfering in receptors signaling. This work highlights the interactions among different components of KKS and contributes to a better understanding of its patho-physiological role.

We compared the synthesis and structural/conformational details of the (66-97) segments of the second transmembrane helix of AT1, MAS and B2, all of which belong to the class of G-protein-coupled receptors (GPCR). Step-by-step monitoring of the coupling reactions during the growth of these transmembrane peptides revealed that the increase in the level of difficulty started at the 6-10 regions of the sequence. Possibly due to their long and hydrophobic sequences, the final estimated synthesis yields decreased progressively by up to 20-25%. Analytical high pressure liquid chromatography showed that the hydrophobicity indexes of each TM-8, -16, -24 and -32 segments correlated linearly with their retention time. Microscopic measurements of peptide-resin beads indicated that, in general, dichloromethane and dimethylsulfoxide were the best solvents for solvating resin beads in the initial and final stages of the synthesis, respectively. Results from electron paramagnetic resonance experiments with Toac (2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) spin-labeled peptide resins revealed that the level of peptide chain mobility throughout the polymer network was in agreement with their swelling data measured in different solvents. Initial results regarding conformational features determined by circular dichroism (CD) spectra revealed typical α-helicoidally structures for MAS and B2 TM32 fragments when in more than roughly 30% (v/v) trifluoroethanol (TFE). In contrast, the AT1-TM32 segment revealed CD spectra, more representatives of a mixture of other secondary helical conformers, regardless of the amount of TFE. These findings observed in different aspects of these receptors' fragments support further investigations of GPCR-type macromolecules.

Background: Amyloidosis is defined as a generic term given to a series of proteins/ polypeptides in the form of amyloid fibrils that are deposited in the tissues and give rise to a set of clinical disorders., Objectives: This work developed an approach to first examine chain association propensities of several amyloidogenic peptides: SNNFGAILSS from the islet amyloid polypeptide (coded IAPP), NAGDVAFV from the protein responsible for corneal amyloidosis (coded Lactoferrin), and (1-42) β-amyloid (coded Amyloid)., Methods: Fmoc-synthesis protocol was applied for the synthesis of IAPP and Lactoferrin whereas Amyloid was synthesized through the Boc-chemistry as early detailed., Results and Conclusion: The fluorescence and light scattering experiments results indicated that Amyloid revealed a surprising reduction in the aggregation process as a function of time (decrease of about 20-30% in 3 days) through both methods. In contrast, the aggregation intensity of IAPP increased around 35% after 3 days via a light scattering procedure. These findings are very relevant for interpretation of the aggregation phenomenon of amyloidogenic peptides. The final part of this work proposed rules for dissolution of aggregated structures based on the Lewis acid and Lewis base properties of solvents. Very low solubility values (6 to 15%) were measured for peptides in water but with increased to around 90% in strong nucleophilic or strong electrophilic organic solvents. However, care should be taken when strong nucleophilic solvents such as DMSO are mixed with the strong electrophilic such as water. Both solvent molecules tend to attract each other rather than to dissolve peptide chains thus lowering the capacity of this type of solution for fibril dissolution., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Obesity is assumed to be a major cause of human essential hypertension; however, the mechanisms responsible for weight-related increase in blood pressure (BP) are not fully understood. The prevalence of hypertension induced by obesity has grown over the years, and the role of the renin-angiotensin-aldosterone system (RAAS) in this process continues to be elucidated. In this scenario, the ob/ob mice are a genetic obesity model generally used for metabolic disorder studies. These mice are normotensive even though they present several metabolic conditions that predispose them to hypertension. Although the normotensive trait in these mice is associated with the poor activation of sympathetic nervous system by the lack of leptin, we demonstrated that ob/ob mice present massively increased aminopeptidase A (APA) activity in the circulation. APA enzyme metabolizes angiotensin (ANG) II into ANG III, a peptide associated with intrarenal angiotensin type 2 (AT 2 ) receptor activation and induction of natriuresis. In these mice, we found increased ANG-III levels in the circulation, high AT 2 receptor expression in the kidney, and enhanced natriuresis. AT 2 receptor blocking and APA inhibition increased BP, suggesting the ANG III-AT 2 receptor axis as a complementary BP control mechanism. Circulating APA activity was significantly reduced by weight loss independently of leptin, indicating the role of fat tissue in APA production. Therefore, in this study we provide new data supporting the role of APA in BP control in ob/ob mouse strain. These findings improve our comprehension about obesity-related hypertension and suggest new tools for its treatment. NEW & NOTEWORTHY In this study, we reported an increased angiotensin III generation in the circulation of ob/ob mice caused by a high aminopeptidase A activity. These findings are associated with an increased natriuresis found in these mice and support the role of renin-angiotensin-aldosterone system as additional mechanism regulating blood pressure in this genetic obese strain., (Copyright © 2017 the American Physiological Society.)

We have developed and validated a fast and sensitive ultra high-performance liquid chromatography with positive ion electrospray ionization tandem mass spectrometry method for determining N-butylscopolamine levels in human plasma using propranolol as an internal standard. The acquisition was set up in the multiple reaction monitoring mode with the transitions m/z 360.3 → 138.0 for N-butylscopolamine and m/z 260.2 → 116.1 for IS. This method uses a liquid-liquid extraction process with dichloromethane. The analyte and IS were chromatographed on a C 18 , 50 × 2.1 mm, 1.7 μm column through isocratic elution with acetonitrile-5 mm ammonium acetate (adjusted to pH 3.0 with formic acid). The method was linear in the 1-1000 pg/mL range for N-butylscopolamine and was selective, precise, accurate and robust. The validated method was successfully applied to perform a bioequivalence study of the reference (Buscopan ® , from Boehringer Ingelheim) and the test sample coated-tablet formulations (from Foundation for Popular Remedy), both containing 10 mg of N-butylscopolamine bromide administered as a single dose. Using 58 healthy volunteers and accounting for the high intra-individual variability confirmed by statistical calculations (38%), the two formulations were considered bioequivalent because the rate and extent of absorption (within 80-125% interval), satisfying international requirements., (Copyright © 2016 John Wiley & Sons, Ltd.)

A large number of antimicrobial peptides (AMPs) acts with high selectivity and specificity through interactions with membrane lipid components. These peptides undergo complex conformational changes in solution; upon binding to an interface, one major conformation is stabilized. Here we describe a study of the interaction between tritrpticin (TRP3), a cathelicidin AMP, and micelles of different chemical composition. The peptide's structure and dynamics were examined using one-dimensional and two-dimensional NMR. Our data showed that the interaction occurred by conformational selection and the peptide acquired similar structures in all systems studied, despite differences in detergent headgroup charge or dipole orientation. Fluorescence and paramagnetic relaxation enhancement experiments showed that the peptide is located in the interface region and is slightly more deeply inserted in 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-1'-rac-glycerol (LMPG, anionic) than in 1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine (LLPC, zwitterionic) micelles. Moreover, the tilt angle of an assumed helical portion of the peptide is similar in both systems. In previous work we proposed that TRP3 acts by a toroidal pore mechanism. In view of the high hydrophobic core exposure, hydration, and curvature presented by micelles, the conformation of TRP3 in these systems could be related to the peptide's conformation in the toroidal pore., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

This study uses EPR, CD, and fluorescence spectroscopy to examine the structure of bradykinin (BK) analogues attaching the paramagnetic amino acid-type Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 3, 7, and 9. The data were correlated with the potencies in muscle contractile experiments and the substrate properties towards the angiotensin I-converting enzyme (ACE). A study of the biological activities in guinea pig ileum and rat uterus indicated that only Toac 0 -BK partially maintained its native biological potency among the tested peptides. This and its counterpart, Toac 3 -BK, maintained the ability to act as ACE substrates. These results indicate that peptides bearing Toac probe far from the ACE cleavage sites were more susceptible to hydrolysis by ACE. The results also emphasize the existence of a finer control for BK-receptor interaction than for BK binding at the catalytic site of this metallodipetidase. The kinetic kcat/Km values decreased from 202.7 to 38.9μM -1 min -1 for BK and Toac 3 -BK, respectively. EPR, CD, and fluorescence experiments reveal a direct relationship between the structure and activity of these paramagnetic peptides. In contrast to the turn-folded structures of the Toac-internally labeled peptides, more extended conformations were displayed by N- or C-terminally Toac-labeled analogues. Lastly, this work supports the feasibility of monitoring the progress of the ACE-hydrolytic process of Toac-attached peptides by examining time-dependent EPR spectral variations., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Angiotensin II (AngII) and kinins (bradykinin (BK) and des-Arg9-bradykinin (DBK)), are potent agents involved in the maintenance of blood pressure and several biological activities, and their better understanding is important to produce new drugs aimed to control arterial blood pressure. Previous studies on ligand-receptor binding have been based on radioactive methods, which led us to study a new method based on the fluorimetric method. A lanthanide attached to the N-terminal segment of the peptide (AngII, BK and DBK), which produces a time-resolved-fluorescent ligand, was used in a binding test with CHO cells expressing the AT1, AT2, B1 or B2 receptors in comparison with the same cell line tested with the radioactive ligand. Our findings indicated that the non-radioactive method provided a comparable result for the angiotensin receptors. On the other hand, the kinin receptors showed a slight reduction in the binding affinity, probably due to the linkage at the N-terminal segment and/or to the lower biological stability associated to the high temperature (37°C) used for the fluorimetric method, while the radioactive one was at 4°C. We can conclude that a time-resolved fluorescence assay would provide a sensitive method as an alternative tool for receptor studies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bradykinin (BK) is a nonapeptide important for several physiological processes such as vasodilatation, increase in vascular permeability and release of inflammatory mediators. BK performs its actions by coupling to and activating the B2 receptor, a family A G-protein coupled receptor. Using a strategy which allows systematical monitoring of BK R1 and R9 residues and B2 receptor acidic residues Glu5.35(226) and Asp6.58(298), our study aims at clarifying the BK interaction profile with the B2 receptor [receptor residue numbers are normalized according to Ballesteros and Weinstein, Methods Neurosci. 25 (1995), pp. 366-428) followed by receptor sequence numbering in brackets]. N- and C-terminal analogs of BK (-A1, -G1, -K1, -E1 and BK-A9) were tested against wild type B2, Glu5.35(226)Ala and Asp6.58(298)Ala B2 mutant receptors for their affinity and capability to elicit responses by mechanical recordings of isolated mice stomach fundus, measuring intracellular calcium mobilization, and competitive fluorimetric binding assays. BK showed 2- and 15-fold decreased potency for Glu5.35(226) and Asp6.58(298) B2 mutant receptors, respectively. In B2-Glu5.35(226)Ala BK analogs showed milder reduction in evaluated parameters. On the other hand, in the B2-Asp6.58(298)Ala mutant, no N-terminal analog was able to elicit any response. However, the BK-A9 analog presented higher affinity parameters than BK in the latter mutant. These findings provide enough support for defining a novel interaction role of BK-R9 and Asp6.58(298) receptor residues.

Developing functional biointerfaces is important for technological applications. We investigated the interaction and adsorption of the antimicrobial peptide tritrpticin (VRRFPWWWPFLRR, TRP3) onto solid and liquid surfaces and the influence of ions on these processes by several techniques. Surface tension measurements showed that salt addition to TRP3 solution causes a high decrease of surface tension due to the adsorption of TRP3 at air-liquid surface. Ellipsometry studies show the TRP3 adsorption on silicon surfaces forming nanometric films that are able to further interact with liposomes. Contact angle measurements gave insight on the nature of thin film and its roughness. AFM shows the topology of the film on the solid substrates. In addition, those techniques also showed that anions can act as modulators on adsorption phenomena and are correlated with the Hofmeister series. The findings of the current work are relevant for the development of functional interfaces such as biocidal surfaces., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra.

Differently than the 4-methylbenzhydrylamine-resin (MBHAR) which contains a methyl group coupled to the phenylmethylamine-functionalized copoly(styrene-divinilbenzene) structure, alternative resins containing the electron-donating 4-tert-butyl- (BUBHAR) or the electron-withdrawing 2-chloro- (ClBHAR) and 2,4-chloro- (diClBHAR) groups were developed as potential supports for α- carboxamide peptide synthesis. Initially, a time-course investigation of HF cleavage reaction (0 °C) with these resins bearing the vasoconstrictor angiotensin II (AngII, DRVYIHPF) or its Gly(8)-AngII analogue revealed that the peptide- BUBHAR linkage is much more labile than those with ClBHAR or diClBHAR. HF cleavage times of near 2 h or longer than 24 h were needed for complete removal of peptide chains from these two classes of resin, respectively. By including MBHAR and benzhydrylamine-resins (BHAR) in this comparative study, the decreasing order of acid stability of the peptidyl- resin linkage was diClBHAR > ClBHAR > BHAR > MBHAR ~ BUBHAR. The same stability order was observed for the HCl/propionic acid hydrolysis reaction (130°C) with the Phe- or Gly-resins. These findings thus suggest that ClBHAR and diClBHAR are not appropriate for use in peptide synthesis. Nevertheless, these supports could still be tested as stationary phases for affinity chromatography. When placed into more apolar solvents, the beads of all of these resins exhibited a greater swelling (as measured by a microscope) or higher mobility of the polymer matrix (as measured with EPR experiments using spin-labeled beads). Moreover, under the latter approach, BUBHAR displayed a comparatively higher solvation degree than did MBHAR (in DCM, DMF and NMP), with slightly higher peptide synthesis yields as well.

Background: Many types of cancer are associated with polymorphisms of the renin-angiotensin system. Our aim was to assess possible association between single-nucleotide polymorphisms (SNPs) of the angiotensin II receptor types 1 (A168G), and 2 (T1247G and A5235G) with breast cancer., Patients and Methods: 242 participating subjects were genotyped and allocated to case or control groups., Results: Genotype distribution (in %) was: for AGTR1 (A168G): AA, AG, GG = 61, 30, 09 for cases, and 69, 25, 06 for controls (p = 0.55); for AGTR2 (T1247G): TT, TG, GG = 84, 12, 04 for cases, and 81, 17, 02 for controls (p = 0.45); for AGTR2 (A5235G): AA, AG, GG = 32, 67, 01 for cases, and 53, 28, 19 for controls (p < 0.0001). Women carrying genotypes AA/AG in the intronic region of angiotensin II type 2 receptor had an 11-fold higher risk of breast cancer than GG carriers., Conclusions: Many types of cancer have been associated with polymorphisms of the renin-angiotensin system. For SNP A5235G, the GG genotype seems to be protective against breast cancer. The other 2 SNPs showed no association. However, SNPs T1247G and A5235G were associated with at least 1 clinical variable, with G being a predictor of better outcome. The use of SNPs A5235G and T1247G (the latter to a lesser degree) as genetic markers should be considered.

This work developed an alternative approach targeting the evaluation of the aggregation propensity of the (1-42) β-amyloid peptide (Alzheimer's disease) and some segments, either attached to a polymer during their synthesis or when free in solution. The solvation behavior of peptide-resins was gauged by measuring the swelling of beads in a microscope and the degree of chain motion through EPR spectra of previously labeled resins with an amino acid-type probe. In terms of comparative solvent dissociation power towards aggregated structures, the findings revealed greater values of peptide-resin swelling, peptide chain mobility and solubility when in strong electron donor dimethylsulfoxide than in strong electron acceptor trifluoroethanol. Otherwise, the weakest chain-chain disruption power was verified for acetonitrile, an internally neutral solvent in terms of Lewis acid/base properties. In complement, fluorescence and light scattering experiments depicted that the 15-35 region plays an essential role in the amyloid peptide fibril formation capacity.

Bradykinin (BK) and des-Arg(9)-bradykinin (DBK) of kallikrein-kinin system exert its effects mediated by the B2 (B2R) and B1 (B1R) receptors, respectively. It was already shown that the deletion of kinin B1R or of B2R induces upregulation of the remaining receptor subtype. However studies on overexpression of B1R or B2R in transgenic animals have supported the importance of the overexpressed receptor but the expression of another receptor subtype has not been determined. Previous study described a marked vasodilatation and increased susceptibility to endotoxic shock which was associated with increased mortality in response to DBK in thoracic aorta from transgenic rat overexpressing the kinin B1R (TGR(Tie2B1)) exclusively in the endothelium. In another study, mice overexpressing B1R in multiple tissues were shown to present high susceptibility to inflammation and to lipopolysaccharide-induced endotoxic shock. Therefore the role of B2R was investigated in the thoracic aorta isolated from TGR(Tie2B1) rats overexpressing the B1R exclusively in the vascular endothelium. Our findings provided evidence for highly increased expression level of the B2R in the transgenic rats. It was reported that under endotoxic shock, these rats exhibited exaggerated hypotension, bradycardia and mortality. It can be suggested that the high mortality during the pathogenesis of endotoxic shock provoked in the transgenic TGR(Tie2B1) rats could be due to the enhanced expression of B2R associated with the overexpression of the B1R., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT(1)R and BKRB(1) or BKRB(2) G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S-S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac(1)-AngII and Toac(0)-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac(3)-AngII and Toac(3)-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT(1)R and BKRB cyclic constructs based on the structure of the CXCR(4), an α-chemokine GPCR-type receptor.

Mutant forms of kinin B(1) receptor (B(1)R) and analogs of the full agonist des-Arg(9)-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 (Cys(100)-Cys(650)), the N-terminal (N(t)) and the EC3 loop C-terminal (C(t)) segments of angiotensin II (AngII) receptor 1 (AT(1)R) have been identified to form an extracellular site for binding the agonist N(t) segment (Asp(1) and Arg(2) residues). Asp(712) residue at the receptor EC3 loop binds the peptide Arg(2) residue. By homology, a similar site might be considered for DABK binding to B(1)R since this receptor contains the same structural elements for composing the site in AT(1)R, namely the disulfide bond and the EC3 loop Asp(712) residue. DABK, Ala(n)-DABK analogs (n=Ala(1)-, Ala(2)-, Ala(3)-, Ala(4)-, Ala(5)-, Ala(6)-, Ala(7)-, Ala(8)-DABK), and other analogs were selected to binding wild-type, Asp712Ala and Cys100Ser mutated B(1)R receptors. The results obtained suggested that the same bimodal scheme adopted for AngII-AT(1)R system may be applied to DABK binding to B(1)R. The most crucial similarity in the two cases is that the N(t) segments of peptides equally bind to the homologous Asp(712) residue of both AT(1)R and B(1)R extracellular sites. Confirming this preliminary supposition, mutation of residues located at the B(1)R extracellular site as EC3 loop Asp(712) and Cys(100) caused the same modifications in biological assays observed in AT(1)R 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 B(1)R receptors., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The present work aimed at evaluating the radiolysis effect upon a set of peptides, most of them involved in physiological functions. To generate reactive radical species, a Co(60) source (up to 15 kGy) was used for controlled gamma irradiation of some peptide solutions including derivatives attaching the stable free radical Toac (2,2,6,6-tetramethypiperidine-1-oxyl-4-amino-4-carboxylic acid). Regardless of the peptide sequence, a nonlinear and progressive degradation of a total of nine peptides was detected. The results were interpreted in the light of the half-life dose (D(1/2)) parameter which represents the dose necessary for 50% peptide structure degradation. The vasoactive angiotensin II (AngII)'s analogue Ang-(1-7) showed greater stability towards gamma ray radiation than bradykinin (BK), Toac(0)-BK, Pro(4)-BK (D(1/2) around 4 and 2 kGy, respectively) which decreased to about 0.5-1.0 kGy in the case of acetyl-α-melanocyte-stimulating hormone (Ac-α-MSH) and substance P (SP). In terms of peptide structural modifications, the data acquired from different analytical methods suggested a Phe to Tyr (or its ortho and/or meta isomers) transformation as a consequence of the hydroxyl moiety insertion. Noteworthy, this effect seemed to be position-dependent as only Phe located at or near the C-terminal portion seemed to display this transformation. In contrast, Met is comparatively more easily oxidized, thus allowing to conclude that gamma irradiation may induce a complex position and/or sequence-dependent effect on peptides. As previously applied for BK, some irradiated peptides were submitted to their by-products purification, indeed a complementary target of the present approach for development of uncommon analogues for further structure-function investigation.

We examined the interaction of the cationic antimicrobial peptide (AMP) tritrpticin (VRRFPWWWPFLRR, TRP3) with Langmuir monolayers of zwitterionic (dipalmitoyl phosphatidylcholine, DPPC, and dipalmitoyl phosphatidylethanolamine, DPPE) and negatively charged phospholipids (dipalmitoyl phosphatidic acid, DPPA, and dipalmitoyl phosphatidylglycerol, DPPG). Both surface pressure and surface potential isotherms became more expanded upon addition of TRP3 (DPPE~DPPC<

We report the effects of a synthetic peptide designed to act as a nuclear localization signal on the treatment of tuberculosis. The peptide contains 21 amino acid residues with the following specific domains: nuclear localization signal from SV 40T, cationic shuttle sequence, and cysteamide group at the C-terminus. The peptide was complexed with the plasmid DNAhsp65 and incorporated into cationic liposomes, forming a pseudo-ternary complex. The same cationic liposomes, composed of egg chicken L-α-phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, and 1,2-dioleoyl-3-trimethylammonium-propane (2:1:1M), were previously evaluated as a gene carrier for tuberculosis immunization protocols with DNAhsp65. The pseudo-ternary complex presented a controlled size (250 nm), spherical-like shape, and various lamellae in liposomes as evaluated by transmission electron microscopy. An assay of fluorescence probe accessibility confirmed insertion of the peptide/DNA into the liposome structure. Peptide addition conferred no cytotoxicity in vitro, and similar therapeutic effects against tuberculosis were seen with four times less DNA compared with naked DNA treatment. Taken together, the results indicate that the pseudo-ternary complex is a promising gene vaccine for tuberculosis treatment. This work contributes to the development of multifunctional nanostructures in the search for strategies for in vivo DNA delivery., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In this work we examine the interaction between the 13-residue cationic antimicrobial peptide (AMP) tritrpticin (VRRFPWWWPFLRR, TRP3) and model membranes of variable lipid composition. The effect on peptide conformational properties was investigated by means of CD (circular dichroism) and fluorescence spectroscopies. Based on the hypothesis that the antibiotic acts through a mechanism involving toroidal pore formation, and taking into account that models of toroidal pores imply the formation of positive curvature, we used large unilamellar vesicles (LUV) to mimic the initial step of peptide-lipid interaction, when the peptide binds to the bilayer membrane, and micelles to mimic the topology of the pore itself, since these aggregates display positive curvature. In order to more faithfully assess the role of curvature, micelles were prepared with lysophospholipids containing (qualitatively and quantitatively) head groups identical to those of bilayer phospholipids. CD and fluorescence spectra showed that, while TRP3 binds to bilayers only when they carry negatively charged phospholipids, binding to micelles occurs irrespective of surface charge, indicating that electrostatic interactions play a less predominant role in the latter case. Moreover, the conformations acquired by the peptide were independent of lipid composition in both bilayers and micelles. However, the conformations were different in bilayers and in micelles, suggesting that curvature has an influence on the secondary structure acquired by the peptide. Fluorescence data pointed to an interfacial location of TRP3 in both types of aggregates. Nevertheless, experiments with a water soluble fluorescence quencher suggested that the tryptophan residues are more accessible to the quencher in micelles than in bilayers. Thus, we propose that bilayers and micelles can be used as models for the two steps of toroidal pore formation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

We find that a common mutation that increases angiotensin I-converting enzyme activity occurs with higher frequency in male patients suffering from refractory temporal lobe epilepsy. However, in their brains, the activity of the enzyme is downregulated. As an explanation, we surprisingly find that carbamazepine, commonly used to treat epilepsy, is an inhibitor of the enzyme, thus providing a direct link between epilepsy and the renin-angiotensin and kallikrein-kinin systems.

We review work on the paramagnetic amino acid 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, TOAC, and its applications in studies of peptides and peptide synthesis. TOAC was the first spin label probe incorporated in peptides by means of a peptide bond. In view of the rigid character of this cyclic molecule and its attachment to the peptide backbone via a peptide bond, TOAC incorporation has been very useful to analyze backbone dynamics and peptide secondary structure. Many of these studies were performed making use of EPR spectroscopy, but other physical techniques, such as X-ray crystallography, CD, fluorescence, NMR, and FT-IR, have been employed. The use of double-labeled synthetic peptides has allowed the investigation of their secondary structure. A large number of studies have focused on the interaction of peptides, both synthetic and biologically active, with membranes. In the latter case, work has been reported on ligands and fragments of GPCR, host defense peptides, phospholamban, and β-amyloid. EPR studies of macroscopically aligned samples have provided information on the orientation of peptides in membranes. More recent studies have focused on peptide-protein and peptide-nucleic acid interactions. Moreover, TOAC has been shown to be a valuable probe for paramagnetic relaxation enhancement NMR studies of the interaction of labeled peptides with proteins. The growth of the number of TOAC-related publications suggests that this unnatural amino acid will find increasing applications in the future.

Background: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity., Methods: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation., Results: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained., Conclusions: In silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders., (Copyright © 2011 S. Karger AG, Basel.)

Angiotensin (Ang) I-converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system.

The use of very highly substituted resins has been avoided for peptide synthesis due to the aggravation of chain-chain interactions within beads. To better evaluate this problem, a combined solvation-peptide synthesis approach was herein developed taking as models, several peptide-resins and with peptide contents values increasing up to near 85%. Influence of peptide sequence and loading to solvation characteristics of these compounds was observed. Moreover, chain-chain distance and chain concentration within the bead were also calculated in different loaded conditions. Of note, a severe shrinking of beads occurred during the α-amine deprotonation step only when in heavily loaded resins, thus suggesting the need for the modification of the solvent system at this step. Finally, the yields of different syntheses in low and heavily loaded conditions were comparable, thus indicating the feasibility of applying this latter "prohibitive" chemical synthesis protocol. We thought these results might be basically credited to the possibility, without the need of increasing molar excess of reactants, of carrying out the coupling reaction in higher concentration of reactants - near three to seven folds - favored by the use of smaller amount of resin. Additionally, the alteration in the solvent system at the α-amine deprotonation step might be also improving the peptide synthesis when in heavily loaded experimental protocol., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Fabry disease is a multisystem X-linked disorder resulting from α-galactosidase A (α-GalA) gene mutations leading to the accumulation of globotriaosylceramide mainly in endothelium compromising heart, kidney, and brain. In Fabry patients, progressive renal failure is frequently treated with angiotensin I-converting enzyme (ACE) inhibitors. We were interested in the possible interactions between ACE inhibitors therapy and the only causative therapy for Fabry disease, the enzyme replacement therapy (ERT) using recombinant human α-GalA (rhα-GalA). Our results suggest that ACE activity was significantly inhibited in plasma of Fabry patients and the blood pressure level decreased just after ERT (at the end of the rhα-GalA infusion). Interestingly, 2 weeks later, ACE activity was significantly upregulated and the plasma levels of angiotensin II increased in the patients treated with rhα-GalA following the elevations of ACE activity. The same inhibitory effect on ACE activity was also observed in rats after rhα-GalA infusion. Furthermore, ACE activity in CHO cells transfected with the human ACE was inhibited dose and time-dependently by rhα-GalA. In vitro, the incubation of plasma from healthy volunteers with rhα-GalA significantly reduced ACE activity. Finally, rhα-GalA also inhibited ACE activity and released galactose residues from purified rabbit lung ACE dose-dependently. In summary, our results suggest that rhα-GalA interacts with ACE and inhibits its activity, possibly by removing the galactose residues from the enzyme. This modulation might have profound impact on the clinical outcome of Fabry patients treated with rhα-GalA.

Previous research showed that disruption of the Cys(18)-Cys(274) bond in the angiotensin II (AngII) AT₁ receptor mutant (C18S), expressed in CHO cells, causes an increase in the basal activity and attenuation of the maximum response to AngII. In addition, this mutant was mostly intracellularly distributed. Our aim was to investigate whether the intracellular presence of the mutant was due to a constitutive internalization or to a defective maturation of the receptor. The first hypothesis was assessed by pretreating the cells with losartan or [Sar¹Leu⁸]-AngII, specific AT₁ receptor antagonists, a maneuver to revert the receptor internalization. The second hypothesis was tested using calnexin, an endoplasmic reticulum marker. We found that treatment with AT₁ receptor antagonists causes an increase in the binding ability of the mutant to AngII. Furthermore, whereas the maximum effect is increased, it reduces the enhanced basal levels of IP₃. The hypothesis for a lack of maturation of the mutant receptor was ruled out because calnexin was poorly colocalized with the intracellular C18S receptor. Our results suggest that the mutation of the AT₁ receptor leads to a conformational structure similar to that of the active mode of the AT₁ receptor, favoring its internalization in the absence of the agonist.

Leptin is a hormone related to metabolism. It also influences blood pressure, but the mechanisms triggered in this process are not yet elucidated. Angiotensin-I converting enzyme (ACE) regulates cardiovascular functions and recently has been associated with metabolism control and obesity. Here, we used ob/ob mice, a model lacking leptin, to answer the question whether ACE and leptin could interact to influence blood pressure, thereby linking the renin-angiotensin system and obesity. These mice are obese and diabetic but have normal 24 h mean arterial pressure. Our results show that plasma and lung ACE activities as well as ACE mRNA expression were significantly decreased in ob/ob mice. In agreement with these findings, the hypotensive effect produced by enalapril administration was attenuated in the obese mice. Plasma renin, angiotensinogen, angiotensin I, bradykinin, and angiotensin 1-7 were increased, whereas plasma angiotensin II concentration was unchanged in obese mice. Chronic infusion of leptin increased renin activity and angiotensin II concentration in both groups and increased ACE activity in ob/ob mice. Acute leptin infusion restored ACE activity in leptin-deficient mice. Moreover, the effect of an ACE inhibitor on blood pressure was not changed in ob/+ mice during leptin treatment but increased four times in obese mice. In summary, our findings show that the renin-angiotensin system is altered in ob/ob mice, with markedly reduced ACE activity, which suggests a possible connection between the renin-angiotensin system and leptin. These results point to an important interplay between the angiotensinergic and the leptinergic systems, which may play a role in the pathogenesis of obesity, hypertension, and metabolic syndrome.

Introduction: Angiotensin II (AII) is the main active product of the renin angiotensin system. Better known effects of AII are via AT1 receptor (AT1R). Expression of AT1R mutants (L265D and L262D) in CHO cells increased cAMP formation when compared to CHO cells expressing the wild type (WT) AT1R. Morphological transformation of CHO cells transfected with mutants correlated with their increased cAMP formation. DNA synthesis was inhibited in these cells too, indicating that cAMP promotes inhibitory effects on transfected CHO cells growth and causes their morphological change from a tumorigenic phenotype to a non-tumorigenic one., Objectives: To assess the importance of leucine 262 and 265 in determining AT1R structure by means of a comparative structural analysis of two mutant peptides and of a wild-type fragment., Methodology: Three peptides had their conformation compared by circular dichroism (CD): L262D(259-272), L265D(259-272) (mutants) and WT(260-277)., Results: Secondary structures were: beta-turn for WT and L262D and random coil for L265D., Conclusions: Strong correlation was found in the results of biochemical, cellular and structural approaches used to compare WT AT1R to mutant types. Random coil structure of the L265D mutant may be a key point to explain those changes observed in biochemical (binding and signal transduction) and proliferation assays (Correa et al., 2005). beta-Turn formation is an important step during early protein folding and this secondary simple structure is present in L262D and WT, but not in L265D. Therefore, leucine 265 seems to play a crucial role in determining an entirely functional AT1R., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Binding of angiotensin II (DRVYIHPF, AngII) to its AT(1) receptor can trigger a process known as tachyphylaxis (loss of receptor response owing to repeated agonist stimulation). We propose a two-state binding model for tachyphylaxis where the N-terminal Asp(1) and Arg(2) residues of the peptide are supposed to initially bind to the N-terminal segment (Arg(23)) and to the EC-3 loop (Asp(281)) of an AT(1) molecule, respectively (state 1). Sequentially, a disruption of the salt bond between the AngII Asp(1) beta-carboxyl function and the receptor Arg(23) can occur with release of the peptide N-terminal segment, favoring the binding of the Arg(2) residue to the EC-3 loop (Asp(178,281), state 2). In the present study, we expanded this investigation by assaying pharmacological properties of different AngII analogs in guinea-pig ileum bearing modifications at positions 1 and 2. Most of these peptides were weak agonists but many of them had the ability to induce tachyphylaxis. These findings support the two-state model for tachyphylaxis, but alternative mechanisms were revealed where state 1 was no longer needed, depending on the chemical structure of AngII residue 1. Otherwise, any modification of the wild type AngII Arg(2) residue was deleterious for the tachyphylaxis mechanism.

Previous studies on angiotensin II (AngII) AT(1) receptor function have revealed that the N-terminal residues of AngII may modulate receptor activation by binding at the receptor extracellular site. A remarkable feature of this site is an insertion of 8 amino acids in the middle of the EC-3 loop including the Cys(274) residue that supposedly makes a disulfide bond with N-terminal Cys(18). As demonstrated by assays with Del(267-275)AT(1), the role of the Cys(18)-Cys(274) disulfide bridge is to keep a conformation of the inserted residues that allows a normal binding of the AngII N-terminal residues. C18S AT(1) receptor mutant, supposedly having a dissociated disulfide bridge, but an intact residue insertion, is constitutively activated and can less efficiently bind AngII. Similar results were observed when the S-S disulfide bond was disrupted in (C18S,C274S) AT(1) receptor. The importance of the free N-terminal amino group of Asp(1) and of the Arg(2) guanidino group for the binding of AngII to C18S mutant with EC-3 loop insertion was investigated by means of assays using AngII peptide analogues bearing a single mutation of Asp(1) for Sar(1) or Arg(2) for Lys(2), as ligands. This study showed that like AngII, [Sar(1)]-AngII can bind the C18S mutant receptor with low affinity whereas [Lys(2)]-AngII binding is still more reduced. Interestingly, when (125)I-AngII instead of (3)H-AngII was used, no significant binding of this mutant was observed although wild type AT(1) receptor was shown to bind all AngII analogues.

A new automated SPE-LC-ESI-MS/MS method was developed and validated to quantify venlafaxine in human plasma using fluoxetine as an internal standard. The analytes were automatically extracted from plasma by C18 SPE cartridges, separated on a C8 RP column and analyzed by MS in the multiple reaction-monitoring (MRM) mode. The method has a chromatographic run time of 4.0 min and a linear calibration curve over the range of 0.25-200 ng/mL (r >0.997). The between-run precisions, based on the percent RSD for replicate quality controls (0.75; 80, and 200 ng/mL), were < 8.5% for all concentrations. The between-run accuracies, based on the percent relative error, were < 4.0%. This method was successfully employed in a bioequivalence study of two venlafaxine capsule formulations (test formulation from Eurofarma (Brazil) and Efexor XR, reference formulation, from Wyeth-Whitehall, Brazil) in 48 healthy volunteers of both sexes who received a single 150 mg dose of each formulation. More than 3000 samples were analyzed eliminating the analyst's exposure to hazardous organic solvents normally employed in off-line liquid-liquid extractions. The 90% confidence interval (CI) of the individual ratio geometric mean for Test/Reference was 91.6-103.4% for AUC(0-48 h) and 102.2-112.6% for C(max). Since both 90% CI for AUC(0-48 h) and C(max) were included in the 80-125% interval proposed by the US Food and Drug Administration (FDA) and the Brazilian National Health Surveillance Agency (ANVISA), the test formulation was considered bioequivalent to Efexor XR according to both the rate and extent of absorption.

The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and detergents--negatively charged sodium dodecyl sulfate (SDS) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS)--was examined by means of EPR, CD, and fluorescence. EPR spectra of partially active TOAC1-AII and inactive TOAC3-AII in aqueous solution indicated fast tumbling, the freedom of motion being greater at the N-terminus. Line broadening occurred upon interaction with micelles. Below SDS critical micelle concentration, broader lines indicated complex formation with tighter molecular packing than in micelles. Small changes in hyperfine splittings evinced TOAC location at the micelle-water interface. The interaction with anionic micelles was more effective than with zwitterionic micelles. Peptide-micelle interaction caused fluorescence increase. The TOAC-promoted intramolecular fluorescence quenching was more pronounced for TOAC3-AII because of the proximity between the nitroxide and Tyr4. CD spectra showed that although both AII and TOAC1-AII presented flexible conformations in water, TOAC3-AII displayed conformational restriction because of the TOAC-imposed bend (Schreier et al., Biopolymers 2004, 74, 389). In HPS, conformational changes were observed for the labeled peptides at neutral and basic pH. In SDS, all peptides underwent pH-dependent conformational changes. Although the spectra suggested similar folds for AII and TOAC1-AII, different conformations were acquired by TOAC3-AII. The membrane environment has been hypothesized to shift conformational equilibria so as to stabilize the receptor-bound conformation of ligands. The fact that TOAC3-AII is unable to acquire conformations similar to those of native AII and partially active TOAC1-AII is probably the explanation for its lack of biological activity., (Copyright 2009 Wiley Periodicals, Inc.)

To probe the binding of a peptide agonist to a G-protein coupled receptor in native membranes, the spin-labeled amino acid analogue 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidino-1-oxyl (TOAC) was substituted at either position 4 or 9 within the substance P peptide (RPKPQQFFGLM-NH2), a potent agonist of the neurokinin-1 receptor. The affinity of the 4-TOAC analog is comparable to the native peptide while the affinity of the 9-TOAC derivative is approximately 250-fold lower. Both peptides activate receptor signaling, though the potency of the 9-TOAC peptide is substantially lower. The utility of these modified ligands for reporting conformational dynamics during the neurokinin-1 receptor activation was explored using EPR spectroscopy, which can determine the real-time dynamics of the TOAC nitroxides in solution. While the binding of both the 4-TOAC substance P and 9-TOAC substance P peptides to isolated cell membranes containing the neurokinin-1 receptor is detected, a bound signal for the 9-TOAC peptide is only obtained under conditions that maintain the receptor in its high-affinity binding state. In contrast, 4-TOAC substance P binding is observed by solution EPR under both low- and high-affinity receptor states, with evidence of a more strongly immobilized peptide in the presence of GDP. In addition, to better understand the conformational consequences of TOAC substitution into substance P as it relates to receptor binding and activation, atomistic models for both the 4- and 9-TOAC versions of the peptide were constructed, and the molecular dynamics calculated via simulated annealing to explore the influence of the TOAC substitutions on backbone structure.

Purpose: The vasoactive octapeptide hormone angiotensin II (DRVYIHPF, AngII) was selected as the target of this2investigation, which was aimed at determining the effect of gamma radiation on peptide structure and biological activity., Materials and Methods: Radiation doses ranging from 1-15 kGy were applied to samples of purified AngII., Results: The measured amount of remaining native hormone decreased non-linearly as the gamma radiation dose increases. Amino acid analysis of these irradiated peptide solutions demonstrated similar, simultaneous modifications of Phe8 and His6 residues along with the increase in the radiation dose. This structural variation of the vasoactive peptide closely resembled the decreasing process of the biological potencies of irradiated peptide solutions in rat uterus and guinea pig ileum muscle preparations., Conclusions: These findings suggest that investigating the effect of gamma radiation on small model molecules such as peptides could be of value for further extending this type of study to other physiologically relevant macromolecules such as proteins. Of note, this unique approach could also be useful in generating different types of peptide analogs (after purification) for application in future classical structure-function studies.

In this work, we show that alternagin-C (ALT-C) and ALT-C PEP, a peptide derived from its sequence, were able to induce angiogenesis in wounded rat skin. A spherical cutaneous excision was made in the back of each animal and treated with three different concentrations of ALT-C or ALT-C PEP. After that, the skin was removed and analyzed to verify the presence of new vessels and the expression of growth factors. ALT-C and ALT-C PEP induced the formation of new vessels and modulated the expression of growth factors, mainly VEGF and FGF1. The expression of VEGF increased and it could be detected up to 7 days after injury. FGF1 also significantly increased, but at a lesser extent than VEGF. In conclusion, the present study shows for the first time the stimulation of angiogenesis in an injured tissue by a disintegrin-like protein and that ALT-C may exert this effect by modulating the expression of growth factors.

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.