Preliminary evaluation of the toxicological, antioxidant and antitumor activities promoted by the compounds 2,4-dihydroxy-benzylidene-thiosemicarbazones an in silico, in vitro and in vivo study.

Thiosemicarbazones are promising classes of compounds with antitumor activity. For this study, six 2,4-dihydroxy-benzylidene-thiosemicarbazones compounds were synthesized. These compounds were submitted to different assays in silico, in vitro and in vivo to evaluate the toxicological, antioxidant and antitumor effects. The in silico results were evaluated by the SwissADME and pkCSM platforms and showed that all compounds had good oral bioavailability profiles. The in vitro and in vivo toxicity assays showed that the compounds showed low cytotoxicity against different normal cells and did not promote hemolytic effects. The single dose acute toxicity test (2000 mg/kg) showed that none of the compounds were toxic to mice. In in vitro antioxidant activity assays, the compounds showed moderate to low activity, with PB17 standing out for the ABTS radical capture assay. The in vivo antioxidant activity highlighted the compounds 1, 6 and 8 that promoted a significant increase in the concentration of liver antioxidant enzymes. Finally, all compounds showed promising antitumor activity against different cell lines, especially MCF-7 and DU145 lines, in addition, they inhibited the growth of sarcoma 180 at concentrations lower than 50 mg/kg. These results showed that the evaluated compounds can be considered as potential antitumor agents.

Antibacterial and antibiofilm activities of thiazolidine-2,4-dione and 4-thioxo-thiazolidin-2-one derivatives against multidrug-resistant Staphylococcus aureus clinical isolates.

AIMS: Antimicrobial resistance is one of the highest priorities in global public health with Staphylococcus aureus among the most important microorganisms due to its rapidly evolving antimicrobial resistance. Despite all the efforts of antimicrobial stewardship, research and development of new antimicrobials are still imperative. The thiazolidine ring is considered a privileged structure for the development of new antimicrobials. This study aimed to compare the antibacterial effects of two analogue series of thiazolidine-2,4-dione and 4-thioxo-thiazolidin-2-one against multidrug-resistant Staph. aureus clinical isolates. METHODS AND RESULTS: The derivatives 1a, 2a and 2b exhibited MIC between 1-32 µg ml-1 , with time-to-kill curves showing a bactericidal effect up to 24 h. In the antibiofilm assay, the most active derivatives were able to inhibit about 90% of biofilm formation. The 4-thioxo-thiazolidine-2-one derivatives were more active against planktonic cells, while the thiazolidine-2,4-dione derivatives were able to disrupt about 50% of the preformed biofilm. In the in vivo infection model using Caenorhabditis elegans as a host, the derivatives 1a, 2a and 2b increased nematode survival with a concentration-dependent effect. Exposure of Staph. aureus to the derivatives 2a and 2b induced surface changes and decrease cell size. None of the derivatives was cytotoxic for human peripheral blood mononuclear cells (PBMC) but showed moderate cytotoxicity for L929 fibroblasts. CONCLUSION: The 5-(3,4-dichlorobenzylidene)-4-thioxothiazolidin-2-one (2b) was the most active derivative against Staph. aureus and showed higher selective indices. SIGNIFICANCE AND IMPACT OF THE STUDY: 4-thioxo-thiazolidin-2-one is a promising scaffold for the research and development of new antimicrobial drugs against multidrug-resistant Staph. aureus.

Analyses of the response of carbapenem-resistant Pseudomonas aeruginosa against monotherapy and combined therapy using quantum dots and proteomics.

Carbapenem-resistant P. aeruginosa (CRPA) has become a serious public health problem and the biofilm formation aggravates this problem. The study aimed to evaluate the occurrence of ß-lactamases and quorum sensing (QS) genes in CRPA isolates, analyze production of biofilm, evaluate the response against meropenem (MPM) and∕or polymyxin B (POL B) and its association with azythromicin (AZT) using quantum dots (QDs) and proteomic analysis. Six CRPA isolates were analyzed. ß-lactamases and QS genes were search using specific PCRs and were tested for biofilm production by quantitative technique. A CRPA isolate, containing blaKPC gene and biofilm-producing, was selected to assess its response to therapy using QDs and the MALDI-TOF. The ß-lactamase detected was blaKPC in 66.7% of the isolates. All isolates were biofilm producers and carriers of the QS genes. QDs-MPM conjugates triggered the formation of biofilm and the association with AZT inhibited this effect. Proteomics analysis showed that treatments with MPM or POL B suppressed the expression of the transglycosylase protein, while combined therapy with AZT induced expression of the RpoN protein. Thus, this study shows that the use of fluorescence combined with the proteomics analysis was promising to understand how a CRPA strain reacts to antimicrobial treatment.

In Vitro and In Vivo Wound Healing and Anti-Inflammatory Activities of Babassu Oil (Attalea speciosa Mart. Ex Spreng., Arecaceae).

Babassu (Attalea speciosa Mart. ex Spreng., Arecaceae) is a palm tree endemic to Brazil and found mainly in the borders of Amazon forest, where the harvesting of its fruits is an important source of income for more than 300,000 people. Among the communities of coconut breakers women, babassu oil is used in culinary, as fuel, and mostly as medicinal oil for the treatment of skin wounds and inflammation. This study aimed to evaluate in vitro and in vivo the wound healing effects of babassu oil. In vitro, babassu oil increased the migration of L929 fibroblasts, inhibited the production of nitric oxide by LPS-stimulated peritoneal macrophages, and increased the levels of INF-γ and IL-6 cytokines production. In vivo, babassu oil accelerated the healing process in a full-thickness splinted wound model, by an increase in the fibroblasts number, blood vessels, and collagen deposition in the wounds. The babassu oil also increased the recruitment of inflammatory cells into the wound site and showed an anti-inflammatory effect in a chronic ear edema model, reducing ear thickness, epidermal hyperplasia, and myeloperoxidase activity. Thus, these data corroborate the use of babassu oil in folk medicine as a remedy to treat skin wounds.

Evaluation of gastroprotective and ulcer healing activities of yellow mombin juice from Spondias mombin L.

Spondias mombin L. (yellow mombin) is a tree with a nutritional fruit that is commonly consumed in the North and Northeast of Brazil, as the juice of its pulp is rich in antioxidant compounds. This study aimed to investigate the gastroprotective and ulcer healing activities of yellow mombin juice (YMJ) in Wistar rats, and to elucidate the possible involved mechanisms. Phytochemical characterization of the lyophilized fruit juice was performed by high-performance liquid chromatography (HPLC). The gastroprotective activity of YMJ was investigated in ethanol (25, 50, and 100% YMJ) and indomethacin (100% YMJ) models of acute gastric ulcer in rats. Then, the effect of YMJ on mucus production and gastric secretions, and the involvement of non-protein sulfhydryl groups and prostaglandins in the gastroprotective process were examined. Moreover, the ulcer healing effect of YMJ was investigated in a model of acetic acid-induced chronic ulcer through histological and immunohistochemical analyses. HPLC results identified the presence of epicatechin (7.1 ± 1.6 µg/mL) and quercetin (17.3 ± 2.5 µg/mL) in YMJ. Ethanol-induced gastric lesions were inhibited by YMJ (25, 50, and 100%) by 42.42, 45.09, and 98.21% respectively, and indomethacin-induced lesions were inhibited by YMJ (100%) by 58.96%, compared to control group. Moreover, YMJ reduced gastric content and total acidy by 57.35 and 71.97%, respectively, compared to the control group. Treatment with YMJ also promoted healing of chronic ulcer, regeneration of the gastric mucosa, and restoration of mucus levels in glandular cells, as confirmed by histological analysis. It also increased cellular proliferation, as demonstrated by high reactivity to Ki-67 and bromodeoxyuridine. In conclusion, YMJ was found to possess gastroprotective and ulcer healing activities that are correlated to its antisecretory action. These results support the commercial exploration of YMJ as a functional food.

Anti-Inflammatory Activity of Babassu Oil and Development of a Microemulsion System for Topical Delivery.

Babassu oil extraction is the main income source in nut breakers communities in northeast of Brazil. Among these communities, babassu oil is used for cooking but also medically to treat skin wounds and inflammation, and vulvovaginitis. This study aimed to evaluate the anti-inflammatory activity of babassu oil and develop a microemulsion system with babassu oil for topical delivery. Topical anti-inflammatory activity was evaluated in mice ear edema using PMA, arachidonic acid, ethyl phenylpropiolate, phenol, and capsaicin as phlogistic agents. A microemulsion system was successfully developed using a Span® 80/Kolliphor® EL ratio of 6 : 4 as the surfactant system (S), propylene glycol and water (3 : 1) as the aqueous phase (A), and babassu oil as the oil phase (O), and analyzed through conductivity, SAXS, DSC, TEM, and rheological assays. Babassu oil and lauric acid showed anti-inflammatory activity in mice ear edema, through inhibition of eicosanoid pathway and bioactive amines. The developed formulation (39% A, 12.2% O, and 48.8% S) was classified as a bicontinuous to o/w transition microemulsion that showed a Newtonian profile. The topical anti-inflammatory activity of microemulsified babassu oil was markedly increased. A new delivery system of babassu microemulsion droplet clusters was designed to enhance the therapeutic efficacy of vegetable oil.

Structure-guided discovery of thiazolidine-2,4-dione derivatives as a novel class of Leishmania major pteridine reductase 1 inhibitors.

Leishmania major, as other protozoan parasites, plague human kind since pre-historic times but it remains a worldwide ailment for which the therapeutic arsenal remains scarce. Although L. major is pteridine- and purine-auxotroph, well-established folate biosynthesis inhibitors, such as methotrexate, have poor effect over the parasite survival. The lack of efficiency is related to an alternative biochemical pathway in which pteridine reductase 1 (PTR1) plays a major role. For this reason, this enzyme has been considered a promising target for anti-leishmanial drug development and several inhibitors that share the substrate scaffold have been reported. In order to design a novel class of PTR1 inhibitors, we employed the thiazolidinone ring as a bioisosteric replacement for pteridine/purine ring. Among seven novel thiazolidine-2,4-dione derivatives reported herein, 2d was identified as the most promising lead by thermal shift assays (ΔTm = 11 °C, p = 0,01). Kinetic assays reveal that 2d has IC50 = 44.67 ± 1.74 µM and shows a noncompetitive behavior. This information guided docking studies and molecular dynamics simulations (50 000 ps) that supports 2d putative binding profile (H-bonding to Ser-111 and Leu-66) and shall be useful to design more potent inhibitors.

Effects of Tityus stigmurus (Thorell 1876) (Scorpiones: Buthidae) venom in isolated perfused rat kidneys.

Scorpions belonging to the Tityus genus are of medical interest in Brazil. Among them, Tityus stigmurus is the main scorpion responsible for stings in the Northeast region. After a sting, the scorpion venom distributes rapidly to the organs, reaching the kidneys quickly. However, there are few studies concerning the renal pathophysiology of scorpion poisoning. In this study, we evaluated the effects of T. stigmurus venom (TsV) on renal parameters in isolated rat kidneys. Wistar rats (n = 6), weighing 250-300 g, were perfused with Krebs-Henseleit solution containing 6 g/100 mL bovine serum albumin. TsV at 0.3 and 1.0 µg/mL was tested, and the effects on perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and electrolyte excretion were analyzed. Effects were observed only at TsV concentration of 1.0 µg/mL, which increased PP (controlPP40' = 92.7 ± 1.95; TsVPP40' = 182.0 ± 4.70* mmHg, *p < 0.05), RVR (controlRVR40' = 3.28 ± 0.23 mmHg; TstRVR40' = 6.76 ± 0.45* mmHg, *p < 0.05), UF (controlUF50' = 0.16 ± 0.04; TstUF50' = 0.60 ± 0.10* mL/g/min,*p < 0.05), GFR and electrolyte excretion, with histological changes that indicate renal tubular injury. In conclusion, T. stigmurus venom induces a transient increase in PP with tubular injury, both of which lead to an augmented electrolyte excretion.

Venomics and antivenomics of Bothrops erythromelas from five geographic populations within the Caatinga ecoregion of northeastern Brazil.

The Caatinga lancehead, Bothrops erythromelas, is a medically relevant species, responsible for most of the snakebite accidents in most parts of its distribution range in northeastern Brazil. The spectrum and geographic variability of its venom toxins were investigated applying a venomics approach to venom pools from five geographic areas within the Caatinga ecoregion. Despite its wide habitat, populations of B. erythromelas from Ceará, Pernambuco, Juazeiro, Paraiba, and Ilha de Itaparica exhibit highly conserved venom proteomes. Mirroring their compositional conservation, the five geographic venom pools also showed qualitatively and quantitatively overlapping antivenomic profiles against antivenoms generated in Vital Brazil (BR) and Clodomiro Picado (CR) Institutes, using different venoms in the immunization mixtures. The paraspecificity exhibited by the Brazilian SAB and the Costa Rican BCL antivenoms against venom toxins from B. erythromelas indicates large immunoreactive epitope conservation across genus Bothrops during the last ~14 million years, thus offering promise for the possibility of generating a broad-spectrum bothropic antivenom. Biological Significance Accidental snakebite envenomings represent an important public health hazard in Brazil. Ninety per cent of the yearly estimated 20-30,000 snakebite accidents are caused by species of the Bothrops genus. Bothrops erythromelas, a small, moderately stocky terrestrial venomous snake, is responsible for most of the snakebite accidents in its broad distribution range in the Caatinga, a large ecoregion in northeastern Brazil. To gain a deeper insight into the spectrum of medically important toxins present in the venom of the Caatinga lancehead, we applied a venomics approach to define the proteome and geographic variability of adult B. erythromelas venoms from five geographic regions. Although intraspecific compositional variation between venoms among specimens from different geographic regions has long been appreciated by herpetologists and toxinologists as a general feature of highly adaptable and widely distributed snake species, the five B. erythromelas populations investigated exhibit highly conserved venom proteomes. The overall toxin profile of the Caatinga lancehead's venom explains the local and systemic effects observed in envenomations by B. erythromelas. The five geographic venom pools sampled also showed qualitatively and quantitatively overlapping antivenomic profiles against antivenoms generated using different bothropic venoms in the immunization mixtures. The large immunoreactive epitope conservation across genus Bothrops offers promise for the generation of a broad-spectrum bothropic antivenom.

Solanidane and iminosolanidane alkaloids from Solanum campaniforme.

From the leaves of Solanum campaniforme (Solanaceae), eight solanidane alkaloids were isolated, four of which contain a p-hydroxyphenylethylamine unit. Their structures were established as: 22ß,23ß-epoxy-solanida-1,4-dien-3-one; 22α,23α-epoxy-10-epi-solanida-1,4,9-trien-3-one; 22α,23α-epoxy-solanida-4-en-3-one; 22ß,23ß-epoxy-solanida-4-en-3-one; (E)-N-[8'(4-hydroxyphenyl)ethyl]-22α,23α-epoxy-solanida-1,4,9-trien-3-imine; (E)-N-[8'(4-hydroxyphenyl)ethyl]-22α,23α-epoxy-solanida-1,4-dien-3-imine; (Z)-N-[8'(4-hydroxyphenyl)ethyl]-22α,23α-epoxy-solanida-1,4,9-trien-3-imine and (Z)-N-[8'(4-hydroxyphenyl)ethyl]-22α,23α-epoxy-solanida-1,4-dien-3-imine. All structures were determined using spectroscopic techniques, such as 1D and 2D NMR, and HRESIMS. The cytotoxicity and the antiophidic activities of the alkaloids were evaluated. The alkaloids did not show any cytotoxicity, but inhibited the main toxic actions of Bothrops pauloensis venom.

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

BACKGROUND: Apis 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. RESULTS: AmV 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. CONCLUSIONS: In 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.

Isolation, homology modeling and renal effects of a C-type natriuretic peptide from the venom of the Brazilian yellow scorpion (Tityus serrulatus).

Mammalian natriuretic peptides (NPs) have been extensively investigated for use as therapeutic agents in the treatment of cardiovascular diseases. Here, we describe the isolation, sequencing and tridimensional homology modeling of the first C-type natriuretic peptide isolated from scorpion venom. In addition, its effects on the renal function of rats and on the mRNA expression of natriuretic peptide receptors in the kidneys are delineated. Fractionation of Tityus serrulatus venom using chromatographic techniques yielded a peptide with a molecular mass of 2190.64 Da, which exhibited the pattern of disulfide bridges that is characteristic of a C-type NP (TsNP, T. serrulatus Natriuretic Peptide). In the isolated perfused rat kidney assay, treatment with two concentrations of TsNP (0.03 and 0.1 µg/mL) increased the perfusion pressure, glomerular filtration rate and urinary flow. After 60 min of treatment at both concentrations, the percentages of sodium, potassium and chloride transport were decreased, and the urinary cGMP concentration was elevated. Natriuretic peptide receptor-A (NPR-A) mRNA expression was down regulated in the kidneys treated with both concentrations of TsNP, whereas NPR-B, NPR-C and CG-C mRNAs were up regulated at the 0.1 µg/mL concentration. In conclusion, this work describes the isolation and modeling of the first natriuretic peptide isolated from scorpion venom. In addition, examinations of the renal actions of TsNP indicate that its effects may be related to the activation of NPR-B, NPR-C and GC-C.

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

Background : Apis 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. Results : AmV and the complex PLA2 + melittin (0.1-50 g/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 M), verapamil (10 M), losartan (100 M), and U-73122 (10 M, a phospholipase C inhibitor), eliminated the AmV-induced contractile effects. Conclusions : In conclusion, AmV caused contractile effect in aorta rings probably through the involvement of voltage-operated calcium channels, AT1 and -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.

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

Background : Apis 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. Results : AmV 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. Conclusions : In 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.(AU)

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

BACKGROUND: Harpalycin 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. METHODS: HP-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. RESULTS: HP-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. CONCLUSION: HP-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.

Inhibition of Neurotoxic Secretory Phospholipases A(2) Enzymatic, Edematogenic, and Myotoxic Activities by Harpalycin 2, an Isoflavone Isolated from Harpalyce brasiliana Benth.

Secretory phospholipases A(2) (sPLA(2)) exert proinflammatory actions through lipid mediators. These enzymes have been found to be elevated in many inflammatory disorders such as rheumatoid arthritis, sepsis, and atherosclerosis. The aim of this study was to evaluate the effect of harpalycin 2 (Har2), an isoflavone isolated from Harpalyce brasiliana Benth., in the enzymatic, edematogenic, and myotoxic activities of sPLA(2) from Bothrops pirajai, Crotalus durissus terrificus, Apis mellifera, and Naja naja venoms. Har2 inhibits all sPLA(2) tested. PrTX-III (B. pirajai venom) was inhibited at about 58.7%, Cdt F15 (C. d. terrificus venom) at 78.8%, Apis (from bee venom) at 87.7%, and Naja (N. naja venom) at 88.1%. Edema induced by exogenous sPLA(2) administration performed in mice paws showed significant inhibition by Har2 at the initial step. In addition, Har2 also inhibited the myotoxic activity of these sPLA(2)s. In order to understand how Har2 interacts with these enzymes, docking calculations were made, indicating that the residues His48 and Asp49 in the active site of these enzymes interacted powerfully with Har2 through hydrogen bonds. These data pointed to a possible anti-inflammatory activity of Har2 through sPLA(2) inhibition.

In vitro studies on Bothrops venoms cytotoxic effect on tumor cells.

BACKGROUND: Animal venoms are complex mixtures of proteins and non proteins components with several biological activities. Snake venoms represent an essentially unexplored source of bioactive compounds that may cure disease conditions which do not respond to currently available therapies. These venoms possess many pharmacological activities, as cytotoxic and/or lytic effects on tumor cells in vitro. Herein, were investigated the in vitro cytotoxicity of three Bothrops venoms in tumor cell lines. METHODS: Cytotoxic effect was evaluated in HCT-8 (colon - human), SF-295 (nervous system - human), HL-60 (human leukemia) and MDAMB-435 (breast - human). Cell density and membrane integrity were determined by the exclusion of propidium iodide. To determine whether Bothrops venoms treated cells were undergoing an apoptotic and/ or necrosis death, phosphatidylserine (PS) externalization was measured after the incubation with the venom. RESULTS: Botrhops venons showed significant cytotoxcity against all cell lines in study. Cell density and membrane integrity were determined by the exclusion of propidium iodide. The Bothrops venoms reduced the cell number and revealed the presence of a necrotic population when the cells was exposed to the B. pauloensis B. diporus and B. pirajai venoms. To determine whether Bothrops venoms treated cells were undergoing an apoptotic and/or necrosis death, PS externalization was measured after the incubation with the venom and it was observed necrotic and apoptotic cells. CONCLUSIONS: All Bothrops venoms tested showed cytotoxicity against tumor cell lines through inducing of necrosis and apoptosis.

Purification and characterization of the biological effects of phospholipase A(2) 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.