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Reck (REversion-inducing Cysteine-rich protein with Kazal motifs) tumor suppressor gene encodes a multifunctional glycoprotein which inhibits the activity of several matrix metalloproteinases (MMPs), and has the ability to modulate the Notch and canonical Wnt pathways. Reck-deficient neuro-progenitor cells undergo precocious differentiation; however, modulation of Reck expression during progression of the neuronal differentiation process is yet to be characterized. In the present study, we demonstrate that Reck expression levels are increased during in vitro neuronal differentiation of PC12 pheochromocytoma cells and P19 murine teratocarcinoma cells and characterize mouse Reck promoter activity during this process. Increased Reck promoter activity was found upon induction of differentiation in PC12 cells, in accordance with its increased mRNA expression levels in mouse in vitro models. Interestingly, Reck overexpression, prior to the beginning of the differentiation protocol, led to diminished efficiency of the neuronal differentiation process. Taken together, our findings suggest that increased Reck expression at early stages of differentiation diminishes the number of neuron-like cells, which are positive for the beta-3 tubulin marker. Our data highlight the importance of Reck expression evaluation to optimize in vitro neuronal differentiation protocols.

Reck (REversion-inducing Cysteine-rich protein with Kazal motifs) tumor suppressor gene encodes a multifunctional glycoprotein which inhibits the activity of several matrix metalloproteinases (MMPs), and has the ability to modulate the Notch and canonical Wnt pathways. Reck-deficient neuro-progenitor cells undergo precocious differentiation; however, modulation of Reck expression during progression of the neuronal differentiation process is yet to be characterized. In the present study, we demonstrate that Reck expression levels are increased during in vitro neuronal differentiation of PC12 pheochromocytoma cells and P19 murine teratocarcinoma cells and characterize mouse Reck promoter activity during this process. Increased Reck promoter activity was found upon induction of differentiation in PC12 cells, in accordance with its increased mRNA expression levels in mouse in vitro models. Interestingly, Reck overexpression, prior to the beginning of the differentiation protocol, led to diminished efficiency of the neuronal differentiation process. Taken together, our findings suggest that increased Reck expression at early stages of differentiation diminishes the number of neuron-like cells, which are positive for the beta-3 tubulin marker. Our data highlight the importance of Reck expression evaluation to optimize in vitro neuronal differentiation protocols.

Bradykinin-potentiating peptides from Bothrops jararaca (Bj) discovered in the early 1960s, were the first natural inhibitors of the angiotensin-converting enzyme (ACE). These peptides belong to a large family of snake venom proline-rich oligopeptides (PROs). One of these peptides, Bj-PRO-9a, was essential for defining ACE as effective drug target and development of captopril, an active site-directed inhibitor of ACE used worldwide for the treatment of human arterial hypertension. Recent experimental evidences demonstrated that cardiovascular effects exerted by different Bj-PROs are due to distinct mechanisms besides of ACE inhibition. In the present work, we have investigated the cardiovascular actions of four Bj-PROs, namely Bj-PRO-9a, -11e, -12b and -13a. Bj-PRO-9a acts upon ACE and BK activities to promote blood pressure reduction. Although the others Bj-PROs are also able to inhibit the ACE activity and to potentiate the BK effects, our results indicate that antihypertensive effect evoked by them involve new mechanisms. Bj-PRO-11e and Bj-PRO-12b involves induction of [Ca(2+)]i transients by so far unknown receptor proteins. Moreover, we have suggested argininosuccinate synthetase and M3 muscarinic receptor as targets for cardiovascular effects elicited by Bj-PRO-13a. In summary, the herein reported results provide evidence that Bj-PRO-mediated effects are not restricted to ACE inhibition or potentiation of BK-induced effects and suggest different actions for each peptide for promoting arterial pressure reduction. The present study reveals the complexity of the effects exerted by Bj-PROs for cardiovascular control, opening avenues for the better understanding of blood pressure regulation and for the development of novel therapeutic approaches.

The identification of novel endogenous and exogenous molecules acting in the complex mechanism of regulating the vascular tonus has always been of great interest. The discovery of bradykinin (1949) and the bradykinin-potentiating peptides (1965) had a pivotal influence in the field, respectively, in understanding cardiovascular pathophysiology and in the development of captopril, the first active-site directed inhibitor of angiotensin-converting enzyme, and used worldwide to treat human hypertension. Both discoveries originated from studies of envenoming by the snake Bothrops jararaca. The aim of the present article is to reveal that the snake proline-rich oligopeptides, known as bradykinin-potentiating peptides, are still a source of surprising scientific discoveries, some of them useful not only to reveal potential new targets but also to introduce prospective lead molecules for drug development. In particular, we emphasize argininosuccinate synthetase as a new functional target for one of bradykinin-potentiating peptides found in B. jararaca, Bj-BPP-10c. This decapeptide leads to argininosuccinate synthetase activation, consequently sustaining increased nitric oxide production, a critical endogenous molecule to reduce the arterial blood pressure.

Background: The bradykinin potentiating peptides (BPPs) are oligopeptides found in different animal venoms. BPPs isolated from Bothrops jararaca venom were the first natural inhibitors described for somatic angiotensin I-converting enzyme (ACE). They were used in the structural modeling for captopril development, a classical ACE inhibitor widely used to treat human hypertension. Methods: We evaluated the effect of BPP-5a on cardiovascular parameters of conscious Wistar (WTs) and spontaneously hypertensive rats (SHRs). Results: In SHR, BPP-5a showed potent cardiovascular effects, at doses ranging from 0.47 to 710 nmol/kg. The maximal changes in mean arterial pressure (MAP) and heart rate (HR) were found at the dose of 2.37 nmol/kg (Δ MAP: −38 ± 4 mmHg, p < 0.01; Δ HR: −71 ± 17 bpm, p < 0.05). Reductions in MAP and HR occurred throughout 6 hours of post-injection period. In contrast to active site-directed ACE inhibitors, no ACE inhibition, evaluated by the Ang I pressor effect, or bradykinin potentiation was observed during the antihypertensive effect of the pentapeptide. In vitro assays showed no effects of BPP-5a upon argininosuccinate synthetase and B1, B2, AT1, AT2 or Mas receptors. Ex vivo assays showed that BPP-5a induced endothelium-dependent vasorelaxation in isolated aortic rings of SHRs and WTs. Conclusions: Although the BPP-5a is considered an ACE inhibitor, our results indicate that its antihypertensive effect is exerted via a unique target, a nitric-oxide-dependent mechanism.

The biological activity of the proline-rich decapeptide Bj-PRO-10c, a processing product of the C-type natriuretic peptide precursor protein, expressed in the brain and the venom gland of the pit viper Bothrops jararaca, was originally attributed to the inhibition of the somatic angiotensin-converting enzyme activity with subsequent anti-hypertensive effect. However, recent results suggest broader biological activity may also be involved in the cardiovascular effects of this peptide. Here we show that Bj-PRO-10c enhances and sustains the generation of nitric oxide (NO) by regulating argininosuccinate synthase activity and thereby velocity of the citrulline–NO cycle. Bj-PRO-10c-mediated effects not restricted to the cardiovascular system, since NO production was also induced in cells of astroglial origin. Bj-PRO-10c was internalized by C6 astroglioma cells where it induces NO production and upregulation of the citrulline–NO cycle cells in a dose-dependent fashion. In view of that, astroglial cells function as l -arginine pool for NO production in neighboring neurons, we suggest a regulatory function for Bj-PRO-10c on the metabolism of this gaseous neurotransmitter in the CNS. Moreover, proliferation of astroglial cells was reduced in the presence of Bj-PRO-10c; however, cell death was not induced. Since NO donors have been studied for the treatment of solid cancers, Bj-PRO-10c may serve as structural model for developing drugs to improve the effects of cancer therapy based on the peptide's ability to augment NO production.

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.

Ndel1 plays multiple roles in neuronal development but it is unknown whether its reported cysteine protease activity is important for these processes. Ndel1 is known to be critical for neurite outgrowth in PC12 cells where it works co-operatively in a complex with DISC1 to allow normal neuritogenesis. Through an initial interest in understanding the regulation of the expression of Ndel1 during neuronal differentiation, we have been able to show that Ndel1 expression and enzyme activity is up-regulated during neurite outgrowth in PC12 cells induced to neural differentiation. Heterologous expression of wild-type Ndel1 (Ndel1WT) in PC12 cells increases the percentage of cells bearing neurites in contrast to the catalytically dead mutant, Ndel1C273A, which caused a decrease. Furthermore depletion of endogenous Ndel1 by RNAi decreased neurite outgrowth, which was rescued by transfection of the enzymatically active Ndel1WT, but not by the Ndel1C273A mutant. Together these data support the notion that the endooligopeptidase activity of Ndel1 plays a crucial role in the differentiation process of PC12 cells to neurons. Genetic data and protein interaction with DISC1 might suggest a role for Ndel1 in neuropsychiatirc conditions.

The understanding of complex physiological processes requires information from many different areas of knowledge. To meet this interdisciplinary scenario, the ability of integrating and articulating information is demanded. The difficulty of such approach arises because, more often than not, information is fragmented through under graduation education in Health Sciences. Shifting from a fragmentary and deep view of many topics to joining them horizontally in a global view is not a trivial task for teachers to implement. To attain that objective we proposed a course herein described-Biochemistry of the envenomation response-aimed at integrating previous contents of Health Sciences courses, following international recommendations of interdisciplinary model. The contents were organized by modules with increasing topic complexity. The full understanding of the envenoming pathophysiology of each module would be attained by the integration of knowledge from different disciplines. Active-learning strategy was employed focusing concept map drawing. Evaluation was obtained by a 30-item Likert-type survey answered by ninety students; 84% of the students considered that the number of relations that they were able to establish as seen by concept maps increased throughout the course. Similarly, 98% considered that both the theme and the strategy adopted in the course contributed to develop an interdisciplinary view.

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-de pend ent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, α-methyl-dl-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

Preeclampsia, a pregnancy-specific syndrome characterized by hypertension, proteinuria and edema, is a major cause of fetal and maternal morbidity and mortality especially in developing countries. Bj-PRO-10c, a proline-rich peptide isolated from Bothrops jararaca venom, has been attributed with potent anti-hypertensive effects. Recently, we have shown that Bj-PRO-10c-induced anti-hypertensive actions involved NO production in spontaneous hypertensive rats. Using in vitro studies we now show that Bj-PRO-10c was able to increase NO production in human umbilical vein endothelial cells from hypertensive pregnant women (HUVEC-PE) to levels observed in HUVEC of normotensive women. Moreover, in the presence of the peptide, eNOS expression as well as argininosuccinate synthase activity, the key rate-limiting enzyme of the citrulline-NO cycle, were enhanced. In addition, excessive superoxide production due to NO deficiency, one of the major deleterious effects of the disease, was inhibited by Bj-PRO-10c. Bj-PRO-10c induced intracellular calcium fluxes in both, HUVEC-PE and HUVEC, which, however, led to activation of eNOS expression only in HUVEC-PE. Since Bj-PRO-10c promoted biological effects in HUVEC from patients suffering from the disorder and not in normotensive pregnant women, we hypothesize that Bj-PRO-10c induces its anti-hypertensive effect in mothers with preeclampsia. Such properties may initiate the development of novel therapeutics for treating preeclampsia.

Baroreflex sensitivity is disturbed in many people with cardiovascular diseases such as hypertension. Brain deficiency of nitric oxide (NO), which is synthesized by NO synthase (NOS) in the citrulline-NO cycle (with argininosuccinate synthase (ASS) activity being the rate-limiting step), contributes to impaired baroreflex. We recently showed that a decapeptide isolated from Bothrops jararaca snake venom, denoted Bj-PRO-10c, exerts powerful and sustained antihypertensive activity. Bj-PRO-10c promoted vasodilatation dependent on the positive modulation of ASS activity and NO production in the endothelium, and also acted on the central nervous system, inducing the release of GABA and glutamate, two important neurotransmitters in the regulation of autonomic systems. We evaluated baroreflex function using the regression line obtained by the best-fit points of measured heart rate (HR) and mean arterial pressure (MAP) data from spontaneously hypertensive rats (SHRs) treated with Bj-PRO-10c. We also investigated molecular mechanisms involved in this effect, both in vitro and in vivo. Bj-PRO-10c mediated an increase in baroreflex sensitivity and a decrease in MAP and HR. The effects exerted by the peptide include an increase in the gene expression of endothelial NOS and ASS. Bj-PRO-10c-induced NO production depended on intracellular calcium fluxes and the activation of a G(i/o)-protein-coupled metabotropic receptor. Bj-PRO-10c induced NO production and the gene expression of ASS and endothelial NOS in the brains of SHRs, thereby improving baroreflex sensitivity. Bj-PRO-10c may reveal novel approaches for treating diseases with impaired baroreflex function.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angiotensin I-converting enzyme (ACE). the inhibition of ACE by the decapeptide Bj-PRO-10c (

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-de pend ent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, alpha-methyl-dl-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

The snake venom proline-rich peptide BPP 10c is an active somatic angiotensin-converting enzyme (sACE) inhibitors. Recently we demonstrated that the anti-hypertensive effect of BPP 10c is not related to the inhibition of sACE alone, thus suggesting that this enzyme is not its only target for blood pressure reduction. In the present work, a biodistribution study in Swiss mice of [125I]-BPP 10c in the absence or in the presence of a saturating concentration of captopril, a selective active-site inhibitor of sACE, demonstrated that: (1) [125I]-BPP 10c was present in several organs and the renal absorption was significantly high; (2) [125I]-BPP 10c showed a clear preference for the kidney, maintaining a high concentration in this organ in the presence of captopril for at least 3 h; (3) The residual amount of [125I]-BPP 10c in the kidney of animals simultaneously treated with captopril suggest that the peptide can interact with other targets different from sACE in this organ. We also showed that Cy3-labeled BPP 10c was internalized by human embryonic kidney cells (HEK-293T). Taken together, these results suggest that sACE inhibition by captopril affects the tissue distribution of [125I]-BPP 10c and that the anti-hypertensive effects of BPP 10c are not only dependent on sACE inhibition.

NUDEL-oligopeptidase is a cytosolic cysteine peptidase, active towards oligopeptides and involved in the conversion and inactivation of a number of bioactive peptides. This protein interacts with neuronal proteins and is essential for brain development and cortical organization during embryogenesis. In this study, 5′-flanking sequences of the human and rabbit NUDEL-oligopeptidase gene were cloned into the pGL3 reporter gene vector and the promoter activity of the full-length fragment and deletions series was measured in transient transfection assays using two different cell lines, namely, C6 rat glioma and NH15 human neuroblastoma. Overall, a very similar pattern of promoter activity was obtained for both rabbit and human NUDEL-oligopeptidase promoter sequences, and their respective serial deletion constructs upon transient transfection into these cell lines. The only exception was for the longest rabbit upstream sequence that displayed about 1.8-fold higher luciferase expression upon transfection into NH15 neuronal cells than that observed upon transfection into C6 glioma cells. On the other hand, no significant difference was observed for the human longest sequence. These results are in good agreement with the expression pattern of NUDEL-oligopeptidase in human and rabbit tissues.

The use of concept maps on the teaching of sciences has been object of worldwide research with different purposes: to detect the previous knowledge of the students on certain topics or to evaluate learning, among others. Based on Ausubel´s cognitive psychology, concept maps assume that the learning is accomplished by assimilation of new concepts and propositions to the students´ cognitive structure, contributing to establish links between the previous and new knowledge. It is especially interesting on the approach of interdisciplinary issues, as many studied in Biochemistry.The relevance of the use of concept maps on biochemistry learning was evaluated on a thirty-hour undergraduation optional course, with interdisciplinary topics, which are not usually included on introductory Biochemistry courses. The course Biochemistry of Animal Venoms was structured in seven module where the biochemical action mechanisms of the venoms of Crotalus sp (south american rattlesnake), Bothrops sp (jararaca), Loxosceles sp (brown spider), Tityus sp (yellow scorpion), Phoneutria sp (armed spider), Apis mellifera (honey bee) and Latrodectus sp (black widow)were discussed. The students worked in small groups and, at each module, there were (1) an oriented study, guided by questions, texts and schemes, supervised by the teachers, (2) the construction of individual concept maps, where the local and systemic effects of the venoms should be predicted by their biochemical composition and (3) the construction of a new map by the group, incorporating the information of the individual maps. The difficulty level of these tasks was gradually increased throughout the course, with lesser time to carry out the tasks, lesser assistance during the oriented study and even lesser information on the venom effects.The course assessment was given by the number, quality and correction of the concepts relationship present in the concept maps, through a questionnaire and by the teachers’ observation. The concept maps produced exceeded the expectation, being gradually more complex at each module. The answers to the questionnaire, in a 1 to 5 scale, showed that the course answered the students’ expectations (4,3), that the topics chosen were satisfactory (4,4) and that the students recommend the course to colleagues (4,6). Besides, the majority agreed that the concept maps improved the learning of the course content (4,5) and that this methodology should be adopted in other courses and disciplines.