Renata de Sousa Alves

Purification and characterization of the biological effects of phospholipase A2 from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA(2) isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the São Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA(2) proteins (named BcPLA(2)1, BcPLA(2)2 and BcPLA(2)3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA(2)1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA(2)1 showed high amino acid sequence identity with PLA(2) group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA(2) and 1POC_A). In addition, BcPLA(2)1 also showed significant overall homology to bee PLA(2). The enzymatic activity induced by native BcPLA(2)1 (20 microg/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA(2)1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA(2) from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA(2)1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA(2), a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration.

Renal and vascular effects of the natriuretic peptide isolated from Crotalus durissus cascavella venom

Crotalus durissus cascavella is a snake that is usually found in the scrublands of northeast Brazil. The components of its venom may have effects on the vascular and renal systems. Recently, a new bradykinin inhibitory peptide has been identified in the venom of the Crotalinae family. The aim of the present study was to investigate the renal and vascular effects of the natriuretic peptide isolated from the venom of Crotalus durissus cascavella (NP2_Casca). The chromatographic profile showed the fractionation of substances identified as convulxin, gyroxin, crotoxin and crotamine, as well as fractions V and VI. The electrophoretic profile of fraction V consisted of several bands ranging from approximately 6kDa to 13kDa, while fraction VI showed only two main electrophoretic bands with molecular weights of approximately 6 and 14kDa. Reverse-phase chromatography showed that NP2_Casca corresponds to about 18% of fraction VI and that this fraction is the main natriuretic peptide. NP2_Casca was compared to other natriuretic peptides from other sources of snake venom. All amino acid sequences that were compared showed a consensus region of XGCFGX, XLDRIX and XSGLGCX. The group treated with NP2_Casca showed an increase in perfusion pressure, renal vascular resistance, urinary flow and glomerular filtration rate. The percent of total and proximal tubular transport of sodium was reduced significantly after administration of the peptide. The mean arterial pressure showed a dose-dependent decrease after infusion of NP2_Casca, and an increase in nitrite production. In the aortic ring assay, NP2_Casca caused a relaxant effect in endothelium-intact thoracic aortic rings precontracted with phenylephrine in the presence and absence of isatin. NP2_Casca failed to relax the aortic rings precontracted with an isosmotic potassium Krebs-Henseleit solution. In conclusion, the natriuretic peptide isolated from Crotalus durissus cascavella venom produced renal and vascular effects. NP2_Casca reduced total and proximal sodium tubular transport, leading to an increase in sodium excretion, thereby demonstrating a diuretic action. A hypotensive effect was displayed in an arterial pressure assay, with an increase in nitrite production, suggesting a possible vasoactive action.

Inhibitory effects of sertraline in rat isolated perfused kidneys and in isolated ring preparations of rat arteries.

Objectives  Sertraline is often prescribed to patients suffering with end stage renal disease, but its action on kidney has not been investigated. We aimed to investigate the pharmacological action of sertraline on rat kidney with emphasis on the underlying mechanisms involved in the vascular actions of the drug.

Nephroprotective effects of (−)-α-bisabolol against ischemic-reperfusion acute kidney injury

BACKGROUND Ischemia/reperfusion (I/R) in kidney is commonly related to acute kidney injury (AKI), essentially through oxidative stress. (-)-α-Bisabolol is a sesquiterpene isolated from the essential oil of a variety of plants, including chamomile, which has important antioxidant activity. STUDY DESIGN This study intends to evaluate the nephroprotective activity of (-)-α-bisabolol (Bis) in both in vivo and in vitro models of kidney I/R. METHODS Male Wistar rats were submitted to right nephrectomy, followed by ischemia by clamping of the renal artery in the left kidney for 60min. and 48h of reperfusion. The animals were treated orally with Bis (100mg/kg) or vehicle for 24h after reperfusion, and placed in metabolic cages, to evaluate water consumption, diuresis, urinary osmolality, classic biochemical markers and urinary KIM-1 (kidney injury molecule-1). Additionally, the left kidney was collected for histological evaluation and determination of glutathione (GSH) and Thiobarbituric Acid Reactive Substances (TBARS) levels. Tubular epithelial cells LLC-MK2 were used to assess Bis effect on in vitro I/R, by MTT assay. It was performed the cellular respiration tests by flow cytometry: evaluation of the production of cytoplasmic reactive oxygen species by DCFH-DA assay and mitochondrial transmembrane potential analysis with the dye rhodamine 123. RESULTS I/R caused alterations in diuresis, water intake, urinary osmolality, plasmatic creatinine, urea and uric acid, creatinine clearance, proteinuria and microalbuminuria. Treatment with Bis ameliorated all of these parameters. Also, KIM-1 level enhanced by I/R was also diminished in groups treated with Bis. The histological examination showed that Bis attenuated the morphological changes caused by I/R, markedly vascular congestion and intratubular deposits of proteinaceous material. Additionally, Bis was able to reduce the changes observed in TBARS and GSH levels in kidney tissue. In in vitro assay, Bis was capable to partially protect the cell lineage against cell damage induced by I/R. CONCLUSION (-)-α-Bisabolol has a nephroprotective effect in kidney I/R, with antioxidant effect. Moreover, this result seems to be associated to a direct protective effect on tubular epithelia.

Integralidade: desafio pedagógico do PET-Saúde /UFC

Practical activities in primary care have been identified as a fulfilling experience, to the extent that they convene professors, students, health professionals, administrators, and the community, who are challenged to add values, knowledge, and experience based on the interdisciplinary and comprehensive nature of the teaching activities. Progress in this area has been identified in the Educational Program for Health Work at UFC, as an efficient methodological proposal for comprehensive, interdisciplinary learning in undergraduate health courses. The current study reports on the pedagogical strategies that fostered an understanding of primary care as a whole through the development of a methodological approach for grasping the total living experience, as a movement in the construction of formative processes.

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A2 from Bothrops pirajai venom.

BackgroundHarpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A2 are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A2 drugs.MethodsHP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated.ResultsHP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 ± 0.28 μg/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA2 inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic acid and p-BPB. HP-2 previous incubated with the platelets inhibits the aggregation induced by untreated PrTX-III as well as arachidonic acid.ConclusionHP-2 changes the structure of PrTX-III, inhibiting the enzymatic activity of this enzyme. In addition, PrTX-III platelet aggregant activity was inhibited by treatment with HP-2, p-BPB and aristolochic acid, and these results were corroborated by docking scores.

Vasoconstrictor effect of Africanized honeybee (Apis mellifera L.) venom on rat aorta

BackgroundApis mellifera stings are a problem for public health worldwide, particularly in Latin America due to the aggressiveness of its Africanized honeybees. Massive poisoning by A. mellifera venom (AmV) affects mainly the cardiovascular system, and several works have described its actions on heart muscle. Nevertheless, no work on the pharmacological action mechanisms of the AmV in isolated aorta has been reported. Thus, the present work aimed to investigate the actions of AmV and its main fractions, phospholipase A2 (PLA2) and melittin, on isolated aorta rings and a probable action mechanism.ResultsAmV and the complex PLA2 + melittin (0.1-50 μg/mL) caused contraction in endothelium-containing aorta rings, but neither isolated PLA2 nor melittin were able to reproduce the effect. Endothelium removal did not change the maximum vasoconstrictor effect elicited by AmV. Ca2+-free medium, as well as treatment with phentolamine (5 μM), verapamil (10 μM), losartan (100 μM), and U-73122 (10 μM, a phospholipase C inhibitor), eliminated the AmV-induced contractile effects.ConclusionsIn conclusion, AmV caused contractile effect in aorta rings probably through the involvement of voltage-operated calcium channels, AT1 and α-adrenergic receptors via the downstream activation of phospholipase C. The protein complex, PLA2 + melittin, was also able to induce vasoconstriction, whereas the isolated proteins were not.

Renal effects of Bunodosoma caissarum crude extract: Prostaglandin and endothelin involvement

&NA; Sea anemones contain a variety of interesting biologically active compounds, including some potent toxins. PLA2 from Bunodosoma caissarum, a sea anemone endemic in the Brazilian southern coast, has shown renal alterations on isolated kidney. The aim of this study was to evaluate the renal and vascular effects of B. caissarum crude extract (BcE) on isolated perfused kidney and arteriolar mesenteric bed, as well the involvement of prostaglandins and endothelin. BcE did not show any effect on arteriolar mesenteric bed, but increased perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate and decreased the percentage of sodium tubular transport on isolated perfused kidney. Indomethacin blocked the renal effects induced by BcE and tezosentan only partially blocked these effects. These results demonstrate the effects of BcE on kidney in situ, suggesting the involvement of prostaglandins and endothelin. HighlightsBunodosoma caissarum crude extract (BcE) has renal effect.BcE renal effects could be blocked by indomethacin.BcE renal effects could be partially blocked by tezosentan.Prostaglandins and endothelin are probably involved on renal effect of BcE.