Almir S. Martins

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.

Genomic and expression analysis of multiple Sry loci from a single Rattus norvegicus Y chromosome

BackgroundSry is a gene known to be essential for testis determination but is also transcribed in adult male tissues. The laboratory rat, Rattus norvegicus, has multiple Y chromosome copies of Sry while most mammals have only a single copy. DNA sequence comparisons with other rodents with multiple Sry copies are inconsistent in divergence patterns and functionality of the multiple copies. To address hypotheses of divergence, gene conversion and functional constraints, we sequenced Sry loci from a single R. norvegicus Y chromosome from the Spontaneously Hypertensive Rat strain (SHR) and analyzed DNA sequences for homology among copies. Next, to determine whether all copies of Sry are expressed, we developed a modification of the fluorescent marked capillary electrophoresis method to generate three different sized amplification products to identify Sry copies. We applied this fragment analysis method to both genomic DNA and cDNA prepared from mRNA from testis and adrenal gland of adult male rats.ResultsY chromosome fragments were amplified and sequenced using primers that included the entire Sry coding region and flanking sequences. The analysis of these sequences identified six Sry loci on the Y chromosome. These are paralogous copies consistent with a single phylogeny and the divergence between any two copies is less than 2%. All copies have a conserved reading frame and amino acid sequence consistent with function. Fragment analysis of genomic DNA showed close approximations of experimental with predicted values, validating the use of this method to identify proportions of each copy. Using the fragment analysis procedure with cDNA samples showed the Sry copies expressed were significantly different from the genomic distribution (testis p < 0.001, adrenal gland p < 0.001), and the testis and adrenal copy distribution in the transcripts were also significantly different from each other (p < 0.001). Total Sry transcript expression, analyzed by real-time PCR, showed significantly higher levels of Sry in testis than adrenal gland (p, 0.001).ConclusionThe SHR Y chromosome contains at least 6 full length copies of the Sry gene. These copies have a conserved coding region and conserved amino acid sequence. The pattern of divergence is not consistent with gene conversion as the mechanism for this conservation. Expression studies show multiple copies expressed in the adult male testis and adrenal glands, with tissue specific differences in expression patterns. Both the DNA sequence analysis and RNA transcript expression analysis are consistent with more than one copy having function and selection preventing divergence although we have no functional evidence.

Prevalence of equine herpesvirus type 1 latency detected by polymerase chain reaction.

Summary. In this study, an improved polymerase chain reaction (PCR) was used for detection of DNA of latent EHV-1 strains from several sources. Three pairs of oligonucleotide primers spanning fragments of 333 bp, 226 bp and 268 bp of the thymidine kinase (tk) gene, and one primer pair spanning 225 bp of the glycoprotein C (gC) gene were used in specific amplifications. Primers for EHV-4 PCR were also designed. Restriction digests with TaqI confirmed the identity of tk PCR fragments from EHV-1. The sensitivity to detect PCR products was further improved by visualisation in silver-stained acrylamide gels. PCR assays were applied to 267 samples including pools of tissue, peripheral blood leukocytes (PBL) and nasal swabs of archived, farms and abattoir specimens from a total of 116 animals. The EHV-1 DNA was found in 88% of the analysed samples. The prevalence of the EHV-1 latent or persistent form in adult horses was similar to others reports but found higher than previously described in foetuses and young foals. EHV-4 latency was not detected in the Brazilian studied specimens.

Regulation of multiple renin―angiotensin system genes by Sry

Background and objective We demonstrated that the Sry gene complex on the spontaneously hypertensive rat (SHR) Y chromosome is a candidate locus for hypertension that accounts for the SHR Y chromosome blood pressure effect. All rat strains examined to date share six Sry loci, and a seventh Sry locus (Sry3) appears to be unique to SHR male rats. Previously, we showed that Sry1 increased activity of the tyrosine hydroxylase promoter in transfected PC12 cells, and Sry1 delivered to adrenal gland of Wistar–Kyoto (WKY) rats increased blood pressure and sympathetic nervous system activity. The objective of this study was to determine whether renin–angiotensin system genes participate in Sry-mediated effects. Method Sry expression vectors were co-transfected into CHO cells with luciferase reporter constructs containing promoters of angiotensinogen (Agt −1430/+22), renin (Ren −1050/−1), angiotensin-converting enzyme (ACE) (ACE −1677/+21) and ACE2 (ACE2 −1091/+83). Results Sry1, Sry2 and Sry3 differentially upregulated activity of the promoters of angiotensinogen, renin and ACE genes and downregulated ACE2 promoter activity. The largest effect was seen with Sry3, which increased activity of angiotensinogen promoter by 1.7-fold, renin promoter by 1.3-fold, ACE promoter by 2.6-fold and decreased activity of ACE2 promoter by 0.5-fold. The effect of Sry1 on promoter activity was significantly less than that of Sry3. Sry2 activated promoters at a significantly lower level than Sry1 did. The result of either an additive effect of Sry regulation of multiple genes in the renin–angiotensin system or alterations in expression of a single gene could favor increased levels of Ang II and decreased levels of Ang-(1-7). Conclusion These actions of Sry could result in increased blood pressure in males and contribute to sex differences in blood pressure.

Fatores de risco na transmissão do lentivírus caprino pelo sêmen

The objective of this work was to evaluate the presence of the DNA provirus of the caprine lentivirus (LVC) in ejaculates of naturally infected males, and to verify the influence of the wash of the semen as well as the presence of testicle inflammation on the viral load. Eight semen collections of seven soropositive reproducers were accomplished, four before testicle injury and four after injury. Amongst the collections carried out at the same week, in one the ejaculate was washed, to withdraw the plasma seminal, and in the other it was not. The provirus DNA was identified both by Nested polymerase chain reaction technique (Nested PCR) and by the viral isolation. The virus was isolated in 7.1% of the samples. The PCR identified the provirus DNA in 35.7% of all samples, 17.9% in the washed samples and 53.6% of the integral semen samples. The injury of the testicle tends to greater flow of virus for the semen, therefore, before injury, 21.4% of the samples were positive and after-injury, 50%. Risk of transmission of the LVC by semen of goat reproducers is strengthened by the presence of testicle inflammations and the fact that the criopreserved semen contains the LVC in infecting form.

Fenofibrate prevents orotic acid—Induced hepatic steatosis in rats

The experiments performed in this report were designed to investigate the mechanisms involved in the metabolic alterations associated with orotic acid-induced hepatic steatosis and the effect of fenofibrate, a stimulant of peroxisome proliferators-activated receptor alpha (PPARalpha), on these alterations. Male Wistar rats were divided into three experimental groups: 1) fed a balanced diet (C); 2) fed a balanced diet supplemented with 1% orotic acid (OA); 3) fed OA diet containing 100 mg.kg(-1) bw.day(-1) fenofibrate (OA+F), for 9 days. Administration of OA to rats induced significant increase in the hepatic total lipids content, marked microvesicular steatosis and decrease in plasma lipids concentrations compared to control group. Fenofibrate treatment prevented fatty liver induction, caused an additional reduction on plasma lipids concentrations and caused a 40% decrease in the lipogenic rate in adipose tissue. The results also showed a 40% increase in lipoprotein lipase (LPL) activity in adipose tissue from OA treated group and fenofibrate administration induced a 50% decrease in LPL activity. The liver mRNA expression of PPARalpha and ACO (acyl CoA oxidase) were 85% and 68% decreased in OA group when compared to control, respectively. Fenofibrate treatment increased the PPARalpha and ACO expressions whereas the CPT-1 (carnitine palmitoyl transferase-1) expression was not altered. Our results have shown that fenofibrate treatment decreases the hepatic lipid content induced by OA which is mediated by an important increase in fatty acid oxidation consequent to an increase in hepatic mRNA expression of PPARalpha and ACO.

Long-term effect of Helicobacter pylori eradication on plasma homocysteine in elderly patients with cobalamin deficiency

Background:Helicobacter pylori gastritis may lead to impairment of the production of pepsinogen and acid, which are essential to cobalamin absorption. In turn, cobalamin deficiency leads to hyperhomocysteinaemia, a risk factor for cardio and cerebrovascular diseases. Aim: To evaluate the effect of H pylori eradication on plasma homocysteine levels in elderly patients. Patients: Sixty-two H pylori-positive elderly patients with cobalamin deficiency were prospectively studied. Methods: Homocysteine and cobalamin concentrations were determined before, 6 and 12 months after H pylori eradication. Results: Corpus atrophy was observed in a few patients; otherwise, in most of them, the degree of corpus gastritis was moderate to severe. The initial homocysteine mean (SD) levels decreased from 41.0 (27.1) to 21.6 (10.1) μmol/l at the 6 month follow-up (p<0.001) and to 13.1 (3.8) μmol/l 12 months after H pylori eradication (p<0.001). Conversely, initial cobalamin mean levels increased from 145.5 (48.7) pmol/l to 209.8 (87.1) pmol/l and to 271.2 (140.8) pmol/l, 6 and 12 months after treatment, respectively (p<0.001 for both). Although the erythrocyte mean corpuscular volume was within reference intervals, it decreased significantly 6 (p = 0.002) and 12 (p<0.001) months after treatment. Conclusions: The results of the current study demonstrated that the eradication of H pylori in elderly patients with cobalamin deficiency is followed by increasing of cobalamin and decreasing of homocysteine blood levels.

A single step duplex PCR to distinguish Entamoeba histolytica from Entamoeba dispar

In this study, a single-step duplex polymerase chain reaction procedure was developed for rapid, specific and sensitive identification of Entamoeba histolytica and for its diagnostic differentiation from E. dispar. Specific oligonucleotide primers were combined for the amplification of a cysteine proteinase 5 gene target sequence of 242 bp, present only in E. histolytica. Additionally, another oligonucleotide primer pair for both the E. histolytica and E. dispar actin gene target of 300 bp was designed to amplify only from amoebae DNA. The PCR developed was specific and efficiently identified and differentiated these parasites from each other in either cultured parasites or from stool material.

Exercise induces renin-angiotensin system unbalance and high collagen expression in the heart of Mas-deficient mice

The renin-angiotensin system (RAS) is involved in the cardiac and vascular remodeling associated with cardiovascular diseases. Angiotensin (Ang) II/AT(1) axis is known to promote cardiac hypertrophy and collagen deposition. In contrast, Ang-(1-7)/Mas axis opposes Ang II effects in the heart producing anti-trophic and anti-fibrotic effects. Exercise training is known to induce cardiac remodeling with physiological hypertrophy without fibrosis. We hypothesize that cardiac remodeling induced by chronic exercise depends on the action of Ang-(1-7)/Mas axis. Thus, we evaluated the effect of exercise training on collagen deposition and RAS components in the heart of FVB/N mice lacking Mas receptor (Mas-KO). Male wild-type and Mas-KO mice were subjected to a moderate-intense swimming exercise training for 6 weeks. The left ventricle (LV) of the animals was sectioned and submitted to qRT-PCR and histological analysis. Circulating and tissue angiotensin peptides were measured by RIA. Sedentary Mas-KO presented a higher circulating Ang II/Ang-(1-7) ratio and an increased ACE2 expression in the LV. Physical training induced in Mas-KO and WT a similar cardiac hypertrophy accompanied by a pronounced increase in collagen I and III mRNA expression. Trained Mas-KO and trained WT presented increased Ang-(1-7) in the blood. However, only in trained-WT there was an increase in Ang-(1-7) in the LV. In summary, we showed that deletion of Mas in FVB/N mice produced an unbalance in RAS equilibrium increasing Ang II/AT(1) arm and inducing deleterious cardiac effects as deposition of extracellular matrix proteins. These data indicate that Ang-(1-7)/Mas axis is an important counter-regulatory mechanism in physical training mediate cardiac adaptations.

Canine distemper virus induces apoptosis in cervical tumor derived cell lines

Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV) induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi), by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control.