Role of the human chymase (CMA1) in the conversion of big-endothelin-1 to endothelin-1 (1-31)

Abstract : The chymase-dependant pathway responsible for converting Big ET-1 to ET-1 was established in vitro. It has only been recently, in 2009, that our group demonstrated that the conversion of Big ET-1 to ET-1 (1-31) can occur in vivo in mice (Simard et al., 2009), knowing that ET-1 (1-31) is converted to ET-1 via NEP in vivo (Fecteau et al., 2005). In addition, our laboratory demonstrated in 2013 that mMCP-4, the murine analogue of human chymase, produces ET-1 (1-31) from the Big ET-1 precursor (Houde et al. 2013). Thus far, in the literature, there are no specific characterizations of recombinant chymases (human or murine). In fact, the group of Murakami published in 1995 a study characterizing the CMA1 (human chymase) in a chymostatin-dependent fashion, using Angiotensin I as a substrate (Murakami et al., 1995). However, chymostatin is a non-specific inhibitor of chymase. It has been shown that chymostatin can inhibit elastase, an enzyme that can convert Angiotensin I to Angiotensin II (Becari et al., 2005). Based on these observations, the proposed hypothesis in the present study suggests that recombinant as well as extracted CMA1 from LUVA (human mast cell line), in addition to soluble fractions of human aortas, convert Big ET-1 into ET-1 (1-31 ) in a TY-51469 (a chymase-specific inhibitor) sensitive manner. In a second component, we studied the enzyme kinetics of CMA1 with regard to the Big ET-1 and Ang I substrate. The affinity of CMA1 against Big ET-1 was greater compared to Ang I (KM Big ET- 1: 12.55 μM and Ang I: 37.53 μM). However, CMA1 was more effective in cleaving Ang I compared to Big ET-1 (Kcat / KM Big ET-1: 6.57 x 10-5 μM-1.s-1 and Ang I: 1.8 x 10-4 ΜM-1.s- 1). In a third component involving in vivo experiments, the pressor effects of Big ET-1, ET-1 and Ang I were tested in conscious mMCP-4 KO mice compared to wild-type mice. The increase in mean arterial pressure after administration of Big ET-1 was greater in wild-type mice compared to mMCP- 4 KO mice. This effect was not observed after administration of ET-1 and / or Ang I.

Characterisation of RNase Y in H. pylori: a non-essential enzyme involved in the processing of cag-PAI non coding RNA 1 (CncR1) sRNA

The importance of Helicobacter pylori as a human pathogen is underlined by the plethora of diseases it is responsible for. The capacity of H. pylori to adapt to the restricted host-associated environment andto evade the host immune response largely depends on a streamlined signalling network. The peculiar H. pylori small genome size combined with its paucity of transcriptional regulators highlights the relevance of post-transcriptional regulatory mechanisms as small non-coding RNAs (sRNAs). However, among the 8 RNases represented in H. pylori genome, a regulator guiding sRNAs metabolism is still not well studied. We investigated for the first time the physiological role in H. pylori G27 strain of the RNase Y enzyme. In the first line of research we provide a comprehensive characterization of the RNase Y activity by analysing its genomic organization and the factors that orchestrate its expression. Then, based on bioinformatic prediction models, we depict the most relevant determinants of RNase Y function, demonstrating a correlation of both structure and domain organization with orthologues represented in Gram-positive bacteria. To unveil the post-transcriptional regulatory effect exerted by the RNase Y, we compared the transcriptome of an RNase Y knock-out mutant to the parental wild type strain by RNA-seq approach. In the second line of research we characterized the activity of this single strand specific endoribonuclease on cag-PAI non coding RNA 1 (CncR1) sRNA. We found that deletion or inactivation of RNase Y led to the accumulation of a 3’-extended CncR1 (CncR1-L) transcript over time. Moreover, beneath its increased half-life, CncR1-L resembled a CncR1 inactive phenotype. Finally, we focused on the characterization of the in vivo interactome of CncR1. We set up a preliminary MS2-affinity purification coupled with RNA-sequencing (MAPS) approach and we evaluated the enrichment of specific targets, demonstrating the suitability of the technique in the H. pylori G27 strain.

Reduced Insulin Clearance And Lower Insulin-degrading Enzyme Expression In The Liver Might Contribute To The Thrifty Phenotype Of Protein-restricted Mice.

Nutrient restriction during the early stages of life usually leads to alterations in glucose homeostasis, mainly insulin secretion and sensitivity, increasing the risk of metabolic disorders in adulthood. Despite growing evidence regarding the importance of insulin clearance during glucose homeostasis in health and disease, no information exists about this process in malnourished animals. Thus, in the present study, we aimed to determine the effect of a nutrient-restricted diet on insulin clearance using a model in which 30-d-old C57BL/6 mice were exposed to a protein-restricted diet for 14 weeks. After this period, we evaluated many metabolic variables and extracted pancreatic islet, liver, gastrocnemius muscle (GCK) and white adipose tissue samples from the control (normal-protein diet) and restricted (low-protein diet, LP) mice. Insulin concentrations were determined using RIA and protein expression and phosphorylation by Western blot analysis. The LP mice exhibited lower body weight, glycaemia, and insulinaemia, increased glucose tolerance and altered insulin dynamics after the glucose challenge. The improved glucose tolerance could partially be explained by an increase in insulin sensitivity through the phosphorylation of the insulin receptor/protein kinase B and AMP-activated protein kinase/acetyl-CoA carboxylase in the liver, whereas the changes in insulin dynamics could be attributed to reduced insulin secretion coupled with reduced insulin clearance and lower insulin-degrading enzyme (IDE) expression in the liver and GCK. In summary, protein-restricted mice not only produce and secrete less insulin, but also remove and degrade less insulin. This phenomenon has the double benefit of sparing insulin while prolonging and potentiating its effects, probably due to the lower expression of IDE in the liver, possibly with long-term consequences.

The Characterization Of The Endoglucanase Cel12a From Gloeophyllum Trabeum Reveals An Enzyme Highly Active On β-glucan.

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50 °C on β-glucan. Under these conditions specific activity was 239.2 ± 9.1 U mg(-1) and the half-life of the enzyme was 84.6 ± 3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the Km was 3.2 ± 0.5 mg mL(-1) and the Vmax was 0.41 ± 0.02 µmol min(-1). Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.

Applications Of Visual Evoked Potentials And Fourier-domain Optical Coherence Tomography In Parkinson's Disease: A Controlled Study.

The goal of this cross-sectional observational study was to quantify the pattern-shift visual evoked potentials (VEP) and the thickness as well as the volume of retinal layers using optical coherence tomography (OCT) across a cohort of Parkinson's disease (PD) patients and age-matched controls. Forty-three PD patients and 38 controls were enrolled. All participants underwent a detailed neurological and ophthalmologic evaluation. Idiopathic PD cases were included. Cases with glaucoma or increased intra-ocular pressure were excluded. Patients were assessed by VEP and high-resolution Fourier-domain OCT, which quantified the inner and outer thicknesses of the retinal layers. VEP latencies and the thicknesses of the retinal layers were the main outcome measures. The mean age, with standard deviation (SD), of the PD patients and controls were 63.1 (7.5) and 62.4 (7.2) years, respectively. The patients were predominantly in the initial Hoehn-Yahr (HY) disease stages (34.8% in stage 1 or 1.5, and 55.8 % in stage 2). The VEP latencies and the thicknesses as well as the volumes of the retinal inner and outer layers of the groups were similar. A negative correlation between the retinal thickness and the age was noted in both groups. The thickness of the retinal nerve fibre layer (RNFL) was 102.7 μm in PD patients vs. 104.2 μm in controls. The thicknesses of retinal layers, VEP, and RNFL of PD patients were similar to those of the controls. Despite the use of a representative cohort of PD patients and high-resolution OCT in this study, further studies are required to establish the validity of using OCT and VEP measurements as the anatomic and functional biomarkers for the evaluation of retinal and visual pathways in PD patients.

Effects Of Misalignments In The Retinal Nerve Fiber Layer Thickness Measurements With Spectral Domain Optical Coherence Tomography.

Purpose. To investigate misalignments (MAs) on retinal nerve fiber layer thickness (RNFLT) measurements obtained with Cirrus(©) SD-OCT. Methods. This was a retrospective, observational, cross-sectional study. Twenty-seven healthy and 29 glaucomatous eyes of 56 individuals with one normal exam and another showing MA were included. MAs were defined as an improper alignment of vertical vessels in the en face image. MAs were classified in complete MA (CMA) and partial MA (PMA), according to their site: 1 (superior, outside the measurement ring (MR)), 2 (superior, within MR), 3 (inferior, within MR), and 4 (inferior, outside MR). We compared RNFLT measurements of aligned versus misaligned exams in all 4 sectors, in the superior area (sectors 1 + 2), inferior area (sectors 3 + 4), and within the measurement ring (sectors 2 + 3). Results. RNFLT measurements at 12 clock-hour of eyes with MAs in the superior area (sectors 1 + 2) were significantly lower than those obtained in the same eyes without MAs (P = 0.043). No significant difference was found in other areas (sectors 1 + 2 + 3 + 4, sectors 3 + 4, and sectors 2 + 3). Conclusion. SD-OCT scans with superior MAs may present lower superior RNFLT measurements compared to aligned exams.

Human Mitochondrial Hsp70 (mortalin): Shedding Light On Atpase Activity, Interaction With Adenosine Nucleotides, Solution Structure And Domain Organization.

The human mitochondrial Hsp70, also called mortalin, is of considerable importance for mitochondria biogenesis and the correct functioning of the cell machinery. In the mitochondrial matrix, mortalin acts in the importing and folding process of nucleus-encoded proteins. The in vivo deregulation of mortalin expression and/or function has been correlated with age-related diseases and certain cancers due to its interaction with the p53 protein. In spite of its critical biological roles, structural and functional studies on mortalin are limited by its insoluble recombinant production. This study provides the first report of the production of folded and soluble recombinant mortalin when co-expressed with the human Hsp70-escort protein 1, but it is still likely prone to self-association. The monomeric fraction of mortalin presented a slightly elongated shape and basal ATPase activity that is higher than that of its cytoplasmic counterpart Hsp70-1A, suggesting that it was obtained in the functional state. Through small angle X-ray scattering, we assessed the low-resolution structural model of monomeric mortalin that is characterized by an elongated shape. This model adequately accommodated high resolution structures of Hsp70 domains indicating its quality. We also observed that mortalin interacts with adenosine nucleotides with high affinity. Thermally induced unfolding experiments indicated that mortalin is formed by at least two domains and that the transition is sensitive to the presence of adenosine nucleotides and that this process is dependent on the presence of Mg2+ ions. Interestingly, the thermal-induced unfolding assays of mortalin suggested the presence of an aggregation/association event, which was not observed for human Hsp70-1A, and this finding may explain its natural tendency for in vivo aggregation. Our study may contribute to the structural understanding of mortalin as well as to contribute for its recombinant production for antitumor compound screenings.

Immobilization Of Papain On Chitin And Chitosan And Recycling Of Soluble Enzyme For Deflocculation Of Saccharomyces Cerevisiae From Bioethanol Distilleries.

Yeast flocculation (Saccharomyces cerevisiae) is one of the most important problems in fuel ethanol production. Yeast flocculation causes operational difficulties and increase in the ethanol cost. Proteolytic enzymes can solve this problem since it does not depend on these changes. The recycling of soluble papain and the immobilization of this enzyme on chitin or chitosan were studied. Some cross-linking agents were evaluated in the action of proteolytic activity of papain. The glutaraldehyde (0.1-10% w·v(-1)), polyethyleneimine (0.5% v·v(-1)), and tripolyphosphate (1-10% w·v(-1)) inactivated the enzyme in this range, respectively. Glutaraldehyde inhibited all treatments of papain immobilization. The chitosan cross-linked with TPP in 5 h of reaction showed the yield of active immobilized enzyme of 15.7% and 6.07% in chitosan treated with 0.1% PEI. Although these immobilizations have been possible, these levels have not been enough to cause deflocculation of yeast cells. Free enzyme was efficient for yeast deflocculation in dosages of 3 to 4 g·L(-1). Recycling of soluble papain by centrifugation was effective for 14 cycles with yeast suspension in time perfectly compatible to industrial conditions. The reuse of proteases applied after yeast suspension by additional yeast centrifugation could be an alternative to cost reduction of these enzymes.

Chasing Cardiac Physiology And Pathology Down The Camkii Cascade.

Calcium dynamics is central in cardiac physiology, as the key event leading to the excitation-contraction coupling (ECC) and relaxation processes. The primary function of Ca(2+) in the heart is the control of mechanical activity developed by the myofibril contractile apparatus. This key role of Ca(2+) signaling explains the subtle and critical control of important events of ECC and relaxation, such Ca(2+) influx and SR Ca(2+) release and uptake. The multifunctional Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a signaling molecule that regulates a diverse array of proteins involved not only in ECC and relaxation, but also in cell death, transcriptional activation of hypertrophy, inflammation and arrhythmias. CaMKII activity is triggered by an increase in intracellular Ca(2+) levels. This activity can be sustained, creating molecular memory after the decline in Ca(2+) concentration, by autophosphorylation of the enzyme, as well as by oxidation, glycosylation and nitrosylation at different sites of the regulatory domain of the kinase. CaMKII activity is enhanced in several cardiac diseases, altering the signaling pathways by which CaMKII regulates the different fundamental proteins involved in functional and transcriptional cardiac processes. Dysregulation of these pathways constitutes a central mechanism of various cardiac disease phenomena, like apoptosis and necrosis during ischemia/reperfusion injury, digitalis exposure, post-acidosis and heart failure arrhythmias, or cardiac hypertrophy. Here we summarize significant aspects of the molecular physiology of CaMKII and provide a conceptual framework for understanding the role of the CaMKII cascade on Ca(2+) regulation and dysregulation in cardiac health and disease.

Sensitivity and specificity of machine learning classifiers for glaucoma diagnosis using Spectral Domain OCT and standard automated perimetry

PURPOSE: To evaluate the sensitivity and specificity of machine learning classifiers (MLCs) for glaucoma diagnosis using Spectral Domain OCT (SD-OCT) and standard automated perimetry (SAP). METHODS: Observational cross-sectional study. Sixty two glaucoma patients and 48 healthy individuals were included. All patients underwent a complete ophthalmologic examination, achromatic standard automated perimetry (SAP) and retinal nerve fiber layer (RNFL) imaging with SD-OCT (Cirrus HD-OCT; Carl Zeiss Meditec Inc., Dublin, California). Receiver operating characteristic (ROC) curves were obtained for all SD-OCT parameters and global indices of SAP. Subsequently, the following MLCs were tested using parameters from the SD-OCT and SAP: Bagging (BAG), Naive-Bayes (NB), Multilayer Perceptron (MLP), Radial Basis Function (RBF), Random Forest (RAN), Ensemble Selection (ENS), Classification Tree (CTREE), Ada Boost M1(ADA),Support Vector Machine Linear (SVML) and Support Vector Machine Gaussian (SVMG). Areas under the receiver operating characteristic curves (aROC) obtained for isolated SAP and OCT parameters were compared with MLCs using OCT+SAP data. RESULTS: Combining OCT and SAP data, MLCs' aROCs varied from 0.777(CTREE) to 0.946 (RAN).The best OCT+SAP aROC obtained with RAN (0.946) was significantly larger the best single OCT parameter (p<0.05), but was not significantly different from the aROC obtained with the best single SAP parameter (p=0.19). CONCLUSION: Machine learning classifiers trained on OCT and SAP data can successfully discriminate between healthy and glaucomatous eyes. The combination of OCT and SAP measurements improved the diagnostic accuracy compared with OCT data alone.

Análise do perfil de humor e da enzima alfa-amilase salivar em indivíduos fisicamente ativos = : Analysis of profile of mood states and salivary alpha amylase enzyme in physically active individuals

Universidade Estadual de Campinas . Faculdade de Educação Física

Bleaching of melanin in the epidermis of South American fur seal and its application on enzyme immunohistochemistry

The South American fur seal (Arctocephalus australis) is an amphibious marine mammal distributed along the Atlantic and Pacific coasts of South America. The species is well adjusted to different habitats due to the morphology of its fin-like members and due to some adaptations in their integumentary system. Immunohistochemical studies are very important to evaluate the mechanisms of skin adaptation due the differential expression of the antigens present in the tissue depending of the region of the body surface. However, its strongly pigmented (melanin) epidermis prevents the visualization of the immuno-histochemical chromogens markers. In this study a melanin bleaching method was developed aimed to allow the visualization of the chromogens without interfering in the antigen-antibody affinity for immunohistochemistry. The analysis of PCNA (proliferating cell nuclear antigen) index in the epidermis of A. australis by immunohistochemistry with diaminobenzidine (DAB) as chromogen was used to test the method. The bleaching of the melanin allowed to obtain the cell proliferation index in epidermis and to avoid false positive results without affecting the immunohistochemical results.

Renin angiotensin system and cardiac hypertrophy after sinoaortic denervation in rats

OBJECTIVE: The aim of this study was to evaluate the role of angiotensin I, II and 1-7 on left ventricular hypertrophy of Wistar and spontaneously hypertensive rats submitted to sinoaortic denervation. METHODS: Ten weeks after sinoaortic denervation, hemodynamic and morphofunctional parameters were analyzed, and the left ventricle was dissected for biochemical analyses. RESULTS: Hypertensive groups (controls and denervated) showed an increase on mean blood pressure compared with normotensive ones (controls and denervated). Blood pressure variability was higher in denervated groups than in their respective controls. Left ventricular mass and collagen content were increased in the normotensive denervated and in both spontaneously hypertensive groups compared with Wistar controls. Both hypertensive groups presented a higher concentration of angiotensin II than Wistar controls, whereas angiotensin 1-7 concentration was decreased in the hypertensive denervated group in relation to the Wistar groups. There was no difference in angiotensin I concentration among groups. CONCLUSION: Our results suggest that not only blood pressure variability and reduced baroreflex sensitivity but also elevated levels of angiotensin II and a reduced concentration of angiotensin 1-7 may contribute to the development of left ventricular hypertrophy. These data indicate that baroreflex dysfunction associated with changes in the renin angiotensin system may be predictive factors of left ventricular hypertrophy and cardiac failure.

Hemodynamic, morphometric and autonomic patterns in hypertensive rats - renin-angiotensin system modulation

BACKGROUND: Spontaneously hypertensive rats develop left ventricular hypertrophy, increased blood pressure and blood pressure variability, which are important determinants of heart damage, like the activation of renin-angiotensin system. AIMS: To investigate the effects of the time-course of hypertension over 1) hemodynamic and autonomic patterns (blood pressure; blood pressure variability; heart rate); 2) left ventricular hypertrophy; and 3) local and systemic Renin-angiotensin system of the spontaneously hypertensive rats. METHODS: Male spontaneously hypertensive rats were randomized into two groups: young (n=13) and adult (n=12). Hemodynamic signals (blood pressure, heart rate), blood pressure variability (BPV) and spectral analysis of the autonomic components of blood pressure were analyzed. LEFT ventricular hypertrophy was measured by the ratio of LV mass to body weight (mg/g), by myocyte diameter (μm) and by relative fibrosis area (RFA, %). ACE and ACE2 activities were measured by fluorometry (UF/min), and plasma renin activity (PRA) was assessed by a radioimmunoassay (ng/mL/h). Cardiac gene expressions of Agt, Ace and Ace2 were quantified by RT-PCR (AU). RESULTS: The time-course of hypertension in spontaneously hypertensive rats increased BPV and reduced the alpha index in adult spontaneously hypertensive rats. Adult rats showed increases in left ventricular hypertrophy and in RFA. Compared to young spontaneously hypertensive rats, adult spontaneously hypertensive rats had lower cardiac ACE and ACE2 activities, and high levels of PRA. No change was observed in gene expression of Renin-angiotensin system components. CONCLUSIONS: The observed autonomic dysfunction and modulation of Renin-angiotensin system activity are contributing factors to end-organ damage in hypertension and could be interacting. Our findings suggest that the management of hypertensive disease must start before blood pressure reaches the highest stable levels and the consequent established end-organ damage is reached.

Enzyme kinetics, structural analysis and molecular modeling studies on a series of Schistosoma mansoni PNP inhibitors

The enzyme purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive molecular target for the development of novel drugs against schistosomiasis, a neglected tropical disease that affects about 200 million people worldwide. In the present work, enzyme kinetic studies were carried out in order to determine the potency and mechanism of inhibition of a series of SmPNP inhibitors. In addition to the biochemical investigations, crystallographic and molecular modeling studies revealed important molecular features for binding affinity towards the target enzyme, leading to the development of structure-activity relationships (SAR).