Adriano M.C. Pimenta

Discovery and Characterization of Alamandine, a Novel Component of the Renin-Angiotensin System

Rationale: The renin–angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1–7). Objective: To characterize a novel component of the RAS, alamandine. Methods and Results: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1–7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1–7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein–coupled receptor, member D. Binding of alamandine to Mas-related G-protein–coupled receptor, member D is blocked by D-Pro7-angiotensin-(1–7), the Mas-related G-protein–coupled receptor, member D ligand β-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/β-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. Conclusions: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders. # Novelty and Significance {#article-title-32}Rationale: The renin–angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1–7). Objective: To characterize a novel component of the RAS, alamandine. Methods and Results: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1–7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1–7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein–coupled receptor, member D. Binding of alamandine to Mas-related G-protein–coupled receptor, member D is blocked by D-Pro7-angiotensin-(1–7), the Mas-related G-protein–coupled receptor, member D ligand &bgr;-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/&bgr;-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. Conclusions: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders.

Costs of Mediterranean and western dietary patterns in a Spanish cohort and their relationship with prospective weight change.

Background: There is a scarcity of studies evaluating the relationship between food costs and adherence to different food patterns and obesity. Methods: This was a dynamic cohort of Spanish university graduates (n = 17 197 for the cross-sectional baseline assessment and n = 11 195 for the prospective follow-up analyses). Mean age was 38.6 (SD 12.2) years, and 60% of participants were women. A 136-item food frequency questionnaire previously validated in Spain was used. Principal component analysis (PCA) was used to derive dietary patterns. Average cost of food was calculated from official Spanish government data. Self-reported weight was previously validated in the cohort. Body weight was assessed both at baseline and during follow-up. Results: PCA identified two dietary patterns, designated as western and Mediterranean. Participants with the highest scores on the western dietary pattern (fifth quintile vs first quintile) spent less money (–€0.64 (−

Epidemiologia do sobrepeso e da obesidade e seus fatores determinantes em Belo Horizonte (MG), Brasil: estudo transversal de base populacional

0.80) per 1000 kcal (95% CI −€0.68 to −€0.61, p for trend <0.001)) on their daily food costs, whereas the opposite was true for the Mediterranean dietary pattern (+€0.71 (+

Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom

0.90) (95% CI +€0.67 to +€0.74, p for trend <0.001). After adjusting for dietary pattern scores and other potential confounders, higher daily food costs were significantly associated with greater weight gain. Conclusions: These data suggest that a Mediterranean dietary pattern is more expensive to follow than a western dietary pattern. This economic barrier should be considered when counselling patients about following a healthy diet because cost may be a prohibitive factor.

A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis

OBJETIVO: Determinar a prevalencia do sobrepeso e da obesidade e investigar os fatores de risco na populacao maior de 18 anos residente na regiao metropolitana de Belo Horizonte, Brasil. METODOS: Foram analisados dados obtidos pela pesquisa sobre padroes de vida realizada pelo Instituto Brasileiro de Geografia e Estatistica entre 1996 e 1997 a partir de uma amostra probabilistica de 1 105 individuos. Medidas de peso e altura foram realizadas por equipes treinadas. O sobrepeso (indice de massa corporal >25,0 kg/m²) e a obesidade (indice de massa corporal > 30,0 kg/m²) foram definidos como variaveis dependentes. Os potenciais fatores associados foram estudados a partir de analises bivariadas e da tecnica de regressao logistica multivariada. RESULTADOS: A prevalencia do sobrepeso e da obesidade foi de 31,1% (IC95%: 27,3 a 35,2) e 5,7% (IC95%: 4,0 a 8,1) nos homens e 25,9% (IC95%: 22,4 a 29,8) e 14,7% (IC95%: 11,9 a 17,9) nas mulheres. Na analise multivariada, a idade e o estado marital permaneceram como fatores de risco independentes para o sobrepeso, enquanto a idade, o sexo e a escolaridade permaneceram como fatores independentes para a obesidade. A interacao entre o sexo feminino e a alta escolaridade constituiu-se em fator protetor para o sobrepeso (OR = 0,52; IC95%: 0,33 a 0,83), mas nao para a obesidade. As mulheres de baixa escolaridade apresentaram alto risco (OR = 5,95; IC95%: 2,51 a 14,12) de desenvolver obesidade em comparacao aos homens. CONCLUSOES: Os resultados indicam que o sobrepeso e a obesidade podem vir a se tornar um serio problema de saude publica na regiao metropolitana de Belo Horizonte. E importante desenvolver estudos que enfoquem a relacao entre o sobrepeso e a obesidade e variaveis comportamentais, como o fumo e o consumo de alcool.

Tityus serrulatus venom peptidomics: assessing venom peptide diversity.

A proteinase, named BmooMPalpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the Aalpha-chain of fibrinogen first, followed by the Bbeta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMPalpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMPalpha-I activity. Since the BmooMPalpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.

Childhood and young adult overweight/obesity and incidence of depression in the SUN project.

BackgroundBacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats.ResultsAn optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins.ConclusionsComparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far.

Nut consumption and incidence of hypertension: The SUN prospective cohort *

MALDI-TOF-TOF and de novo sequencing were employed to assess the Tityus serrulatus venom peptide diversity. Previous works has shown the cornucopia of molecular masses, ranging from 800 to 3000Da, present in the venom from this and other scorpions species. This work reports the identification/sequencing of several of these peptides. The majority of the peptides found were fragments of larger venom toxins. For instance, 28 peptides could be identified as fragments from Pape proteins, 10 peptides corresponded to N-terminal fragments of the TsK beta (scorpine-like) toxin and fragments of potassium channel toxins (other than the k-beta) were sequenced as well. N-terminal fragments from the T. serrulatus hypotensins-I and II and a novel hypotensin-like peptide could also be found. This work also reports the sequencing of novel peptides without sequence similarities to other known molecules.

Purification, amino-acid sequence and partial characterization of two toxins with anti-insect activity from the venom of the South American scorpion Tityus bahiensis (Buthidae).

This study included 11,825 participants of a Spanish dynamic prospective cohort based on former students from University of Navarra, registered professionals from some Spanish provinces, and university graduates from other associations, followed‐up for 6.1 years. We aimed to assess the association between childhood or young adult overweight/obesity and the risk of depression. Participants were asked to select which of nine figures most closely represented their body shape at ages 5 and 20 years. Childhood and young adult overweight/obesity was defined as those cases in which participants reported body shape corresponding to the figures 6–9 (more obese categories) at age 5 or 20, respectively. A subject was classified as incident case of depression if he/she was initially free of depression and reported physician‐made diagnosis of depression and/or the use of antidepressant medication in at least one of biannual follow‐up questionnaires. The association between childhood and young adult overweight/obesity and incidence of depression was estimated by multiple‐adjusted hazard ratio (HR) and its 95% confidence interval (95% CI). Overweight/obesity at age 5 years predicted an increased risk for adult depression (HR = 1.50, 95% CI = 1.06–2.12), and a stronger association was observed at age 20 years ((HR = 2.22, 95% CI = 1.22–4.08), (subjects younger than 30 years at recruitment were excluded from this last analysis)). Childhood or young adult overweight/obesity was associated with elevated risk of adult depression. These results, if causal and confirmed in other prospective studies, support treating childhood and young adult overweight/obesity as part of comprehensive adult depression prevention efforts.

Animal toxins: state of the art - perspectives in health and biotechnology

BACKGROUND AND AIMS The consumption of tree nuts could reduce the risk of hypertension, but scarce research has been done to evaluate this potential association. We assessed the association between nut consumption and the incidence of hypertension among 9919 Spanish university graduates followed-up biennially for a median of 4.3 years. METHODS AND RESULTS Food habits were assessed with a validated 136-item food-frequency questionnaire. Nut consumption was classified into four categories: rarely/never, 1-3/month, 1/week, and 2+/week. A participant was classified as an incident case of hypertension when, being free of hypertension at baseline, he/she subsequently reported a physician-made diagnosis of hypertension in at least one of the follow-up questionnaires. The incidence of hypertension was 12.4 per 1000 person-years. We found no association between nut consumption and incidence of hypertension after adjusting for sex, age and other dietary and non-dietary potential confounders (hazard ratio [HR] for those in the highest vs. lowest nut consumption category=0.77 [IC 95%: 0.46-1.30] p=0.795). Results were not modified when we stratified them analyses according to sex or to body mass index. CONCLUSION Our data do not provide evidence for an inverse association between nut consumption and incident hypertension in our cohort. Further results from cohorts and trials with a higher baseline risk of hypertension should be obtained to test this relationship.