Inhibitory effects of gallic acid ester derivatives on Saccharomyces cerevisiae multidrug resistance protein Pdr5p.

Overexpression of the Saccharomyces cerevisiae ABC transporter Pdr5p confers resistance to a range of structurally unrelated xenobiotics. This property allows Pdr5p to be used as a target for novel multidrug resistance reversal reagents or chemosensitizers. Herein, we report the effects of gallic acid derivatives with substitutions either on the ester moiety or in the benzene ring on the activity of Pdr5p. Compounds with a longer side chain (8-16 carbons) resulted in greater inhibition of Pdr5p ATPase. Derivatives with side chains of 8-12 carbons that retained hydroxyl groups on the benzene ring extensively inhibited Pdr5p ATPase activity. These compounds almost completely inhibited the efflux of the Pdr5p fluorescent substrate Rhodamine 6G and at 25 muM chemosensitized the Pdr5p-overexpressing strain AD124567 to fluconazole (0.4 mg mL(-1)). Gallic acid derivatives may be a new class of Pdr5p inhibitors.

Hydroxychalcones induce apoptosis in B16-F10 melanoma cells via GSH and ATP depletion.

Searching for leading compounds of new drugs for cancer therapy, we studied the toxicity of 13 hydroxychalcones never tested before toward melanoma cell line (B16-F10). The compounds were obtained by aldolic condensation between aldehydes and hydroxylated acetophenones, in alkaline conditions. Three of them showed cytotoxicity to the cell line. Two of them induced mitochondrial GSH and ATP depletion and promoted cell death through apoptosis in melanoma cells. One of the compounds induced cell death through necrosis but did not significantly decrease the intracellular mitochondrial GSH and ATP levels in melanoma cells. The results suggest that the predominant factor for the activity is the molecule shape, and secondarily the number of hydroxyl groups.

BE-I-PLA2, a novel acidic phospholipase A2 from Bothrops erythromelas venom: isolation, cloning and characterization as potent anti-platelet and inductor of prostaglandin I2 release by endothelial cells.

A novel acidic Asp49 phospholipase A(2) was isolated from Bothrops erythromelas (jararaca malha-de-cascavel) snake venom by four chromatographic steps. BE-I-PLA2 present a molecular weight of 13,649.57 Da as estimated by mass spectrometry. N-terminal and four internal peptides were sequenced, covering around one-third of the complete toxin sequence. The complete BE-I-PLA2 cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 457 bp and encodes a protein with significant sequence similarity to many other phospholipase A(2) from snake venoms. When tested in platelet rich plasma, the enzyme showed a potent inhibitory effect on aggregation induced by arachidonic acid and collagen, but not ADP. On the other hand, BE-I-PLA2 did not modify aggregation in washed platelet. Furthermore, no action of BE-I-PLA2 on the principal platelets receptors was observed. Chemical modification with p-bromophenacyl bromide abolished the enzymatic activity of BE-I-PLA2, but its anti-platelet activity was only partially inhibited. In human umbilical-cord veins endothelial cells, BE-I-PLA2 was neither apoptotic nor proliferative but stimulated endothelial cells to release prostaglandin I(2), suggesting an increase of its potential anti-platelet activity in vivo. Further studies are required in order to determine the exact mechanism of action of BE-I-PLA2 in the inhibition of platelet aggregation.

Releasing or expression modulating mediator involved in hemostasis by Berythractivase and Jararhagin (SVMPs).

PIII snake venom metalloproteases (SVMPs) are structurally related to ADAMs (a disintegrin and metalloprotease human family of proteins). Berythractivase and Jararhagin are PIII SVMPs with 69% homology with different hemostatic properties. In order to clarify these differences and further characterize the biological effects of these proteins, we compared the effect of both proteases on human umbilical vein endothelial cell (HUVEC) for evaluating the release and modulation of coagulation and fibrinolysis mechanisms as well as the expression of their correlated genes. We found that both proteins increase the von Willebrand factor liberation, but did not modulate gene expression. Berythractivase, differently from Jararhagin increased the expression of tissue factor. Our results showed that both SVMPs (Berythractivase and Jararhagin) activate HUVEC releasing or modulating mediators involved in hemostasis. Meanwhile, we can suggest through the up-regulation of TF gene that the studied SVMP acts in a specific manner, suggesting that Jararhagin has preferentially a local action, while Berythractivase can be assumed as a systemic pro-coagulant protein with activity on the surface of HUVECs.

Losac, a factor X activator from Lonomia obliqua bristle extract: its role in the pathophysiological mechanisms and cell survival.

Contact with the bristles of the caterpillar Lonomia obliqua can cause serious hemorrhage. Previously it was reported that a procoagulant protein (Lopap) in the bristle extract of L. obliqua increases cell longevity by inhibiting apoptosis. In this work, we purified from bristle extract a factor X activator that stimulates proliferation of endothelial cells. This protein, named Losac, was purified by ion exchange chromatography, followed by gel filtration chromatography and reverse-phase HPLC. Losac is a 45-kDa protein that activates factor X in a concentration-dependent manner and does not depend on calcium ions. In cultures of HUVECs, Losac increased cell proliferation and inhibited the apoptosis induced by starvation. HUVECs incubated with Losac (0.58microM for 1h) increased release of nitric oxide and tissue-plasminogen activator, which both may mediate anti-apoptosis. Losac also increased slightly the decay-accelerating factor (DAF=CD55), which protects cells from complement-mediated lysis. On the other hand, Losac did not alter the release or expression of von Willebrand factor, tissue factor, intercellular adhesion molecule-1, interleukin-8, and prostacyclin. These characteristics indicate that Losac, a protein with procoagulant activity, also functions as a growth stimulator and an inhibitor of cellular death for endothelial cells. Losac may have biotechnological applications, including the reduction of cell death and consequently increased productivity of animal cell cultures, and the use of hemolymph of L. obliqua for this purpose is already being explored. Further study is required to elucidate the mechanism for the inhibition of apoptosis by Losac.

Losac, a factor X activator from Lonomia obliqua bristle extract Its role in the pathophysiological mechanisms and cell survival

Contact with the bristles of the caterpillar Lonomia obliqua can cause serious hemorrhage. Previously it was reported that a procoagulant protein (Lopap) in the bristle extract of L. obliqua increases cell longevity by inhibiting apoptosis. In this work, we purified from bristle extract a factor X activator that stimulates proliferation of endothelial cells. This protein, named Losac, was purified by ion exchange chromatography, followed by gel filtration chromatography and reverse-phase HPLC. Losac is a 45-kDa protein that activates factor X in a concentration-dependent manner and does not depend on calcium ions. In cultures of HUVECs, Losac increased cell proliferation and inhibited the apoptosis induced by starvation. HUVECs incubated with Losac (0.58 ìM for 1 h) increased release of nitric oxide and tissue-plasminogen activator, which both may mediate anti-apoptosis. Losac also increased slightly the decay-accelerating factor (DAF = CD55), which protects cells from complement-mediated lysis. On the other hand, Losac did not alter the release or expression of von Willebrand factor, tissue factor, intercellular adhesion molecule-1, interleukin-8, and prostacyclin. These characteristics indicate that Losac, a protein with procoagulant activity, also functions as a growth stimulator and an inhibitor of cellular death for endothelial cells. Losac may have biotechnological applications, including the reduction of cell death and consequently increased productivity of animal cell cultures, and the use of hemolymph of L. obliqua for this purpose is already being explored. Further study is required to elucidate the mechanism for the inhibition of apoptosis by Losac.

Insularinase A, a prothrombin activator from Bothrops insularis venom, is a metalloprotease derived from a gene encoding protease and disintegrin domains.

The first low-molecular-mass metalloprotease presenting prothrombin activating activity was purified from Bothrops insularis venom and named insularinase A. It is a single-chain protease with a molecular mass of 22 639 Da. cDNA sequence analysis revealed that the disintegrin domain of the precursor protein is post-translationally processed, producing the mature insularinase A. Analysis of its deduced amino acid sequence showed a high similarity with several fibrin(ogen)olytic metalloproteases and only a moderate similarity with prothrombin activators. However, SDS-PAGE of prothrombin after activation by insularinase A showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin independently of the prothrombinase complex. In addition, insularinase A activates factor X and hydrolyses fibrinogen and fibrin. Chelating agents fully inhibit all insularinase A activities. Insularinase A induced neither detachment nor apoptosis of human endothelial cells and was also not able to trigger an endothelial proinflammatory cell response. Nitric oxide and prostacyclin levels released by endothelial cells were significantly increased after treatment with insularinase A. Our results show that, although its primary structure is related to class P-I fibrin(ogen)olytic metalloproteases, insularinase A is functionally similar to group A prothrombin activators.

A prothrombin activator (Lopap) modulating inflammation, coagulation and cell survival mechanisms.

A severe hemorrhagic syndrome produced by contact with Lonomia obliqua caterpillars has become epidemic in southern Brazil. A significant thrombin production with intense consumption of fibrinogen and high D-dimer production indicates a consumption coagulopathy and secondary fibrinolysis in patients. Lopap is a single-chain 69kDa serine protease isolated from the crude extract of L. obliqua bristles. Experiments in mice showed that the purified protein, similar to the crude extract, causes uncoagulable blood by fibrinogen depletion. In order to characterize the effects of Lopap on cells involved with hemostatic system, we performed experiments using human umbilical vein endothelial cells (HUVECs). Our results show that Lopap exerts a direct effect on endothelial cells by increasing the liberation of molecules involved in the regulation of vascular tone, inhibiting platelet activation and chemotaxis, apart from inducing the expression of cell adhesion molecules which participate in inflammatory responses. The release or new synthesis of mediators involved in coagulation as von Willebrand factor and tissue factor, or in fibrinolysis as tissue plasminogen activator, was not affected by Lopap. Also our results demonstrated that Lopap acts on cell survival of HUVECs, regulating the expression of molecules as NO and avoiding cell death.

The snake venom metalloproteases berythractivase and jararhagin activate endothelial cells.

PIII snake venom metalloproteases (SVMPs) are metalloproteases structurally related to ADAMs (a disintegrin and metalloprotease human family of proteins). Berythractivase and jararhagin are PIII SVMPs with 69% homology that have different hemostatic properties. In order to clarify these differences and further characterize the biological effects of these proteins, we have analyzed the effect of both proteases on human umbilical-vein endothelial cell functions. We found that both proteins enhanced nitric oxide generation, prostacyclin production and interleukin-8 release. Berythractivase but not jararhagin increased the expression of decay accelerating factor. Jararhagin decreased cell viability in a concentration-dependent manner and induced cellular apoptosis, while berythractivase did not modulate cell survival. Our results show for the first time that, besides the known anti-aggregating or procoagulant effects of PIII SVMPs, these proteins trigger endothelial cell effector responses. Although structurally related, berythractivase and jararhagin induce a dissimilar generation and release of endothelial molecules that may account for their different hemorrhagic activity.

Modulação de respostas de células endoteliais (HUVECs) induzidas por proteases fibrino(geno)líticas e ativadores de coagulação, obtidas de venenos animais / Modulation of endothelial cells responses(HUVECs) induced by fibrin(ogen)olytic and coagulation activators proteases, obtained of animal venoms

O contato com as cerdas da lagarta Lonomía oblíqua, que causa uma severa síndrome hemorrágica, tem se tornado uma epidemia no sul do Brasil. A geração de trombina com intenso consumo de fibrinogênio e aumento nos níveis de produto de degradação de fibrina (D-dimer), indicam uma coagulopatia de consumo com ativação secundária da fibrinólise nos pacientes acidentados. o Lopap é uma protease de aproximadamente 69 kDa que foi isolada do extrato bruto das cerdas da L. oblíqua. Experimentos em camundongos mostraram que o Lopap purificado, semelhante ao extrato bruto, causa incoagulabilidade sangüínea por depleção de fibrinogênio. Distúrbios hemostáticos severos também ocorrem em pacientes envenenados por picadas de serpentes. Os venenos de serpentes possuem proteínas que interferem em inúmeros processos biológicos, incluindo hemostasia. Hemorragia e incoagulabilidade sangüínea são distúrbios sistêmicos comumentes observados em pacientes picados por serpentes do gênero Bothrops. As metaloproteases de venenos de serpentes (SVMPs) da classe P-III, são estruturalmente relacionadas com as ADAMs (a disintegrin and metalloprotease human family of proteins) e estão presentes em diferentes venenos de serpentes. A Berythractivase que foi purificada do veneno de B. erythromelas, e a Jararagina purificada do veneno de B. jarara ca, são SVMPs da classe P-III com 69 por cento de similaridade, porém, possuem diferentes propriedades hemostáticas. Para caracterizar o efeito do Lopap, Berythractivase e Jararagina sobre células envolvidas com o sistema hemostático, realizamos experimentos usando células endoteliais de cordão umbilical humano (HUVECs) avaliando a influência das proteases nos mecanismos de coagulação, fibrinólise e inflamatório, e também a expressão de genes envolvidos em ambos os mecanismos. Nossos resultados mostram que o Lopap exerce um efeito direto sobre as células endoteliais, aumentando a liberação de moléculas envolvidas na regulação do tonus vascular, inibição da ativação plaquetária e quimiotaxia, além de induzir a expressão de moléculas de adesão cellular que participam da resposta inflamatória. O aumento na liberação ou na síntese de mediadores envolvidos na coagulação, como o fator de von Willebrand e fator tecidual, ou envolvidos na fibrinólise, como o ativador do plasminogênio tecidual, não foram estimulados pelo Lopap. Nossos resultados também demonstraram que o Lopap aumenta a sobrevida celular das HUVECs, regulando a expressão de moléculas, como óxido nítrico e proteínas da família Bcl-2, evitando a morte celular. Por outro lado, verificamos que a Berythractivase e a Jararagina aumentam a geração de óxido nítrico, a produção de prostaciclina e a liberação de fator de von Willebrand, ativador do plasminogênio tecidual e interleucina-8. A Berythractivase, mas não a Jararagina, aumentou a expressão de DAF (decay accelerating facto r) e fator tecidual. Jararagina diminui a viabilidade e induz a apoptose celular de maneira concentração dependente, entretanto, a Berythractivase não altera a sobrevida celular. Esses resultados mostram que, além do efeito antiagregante ou pró-coagulantes das SVMPs da classe P-III, estas proteínas tem efeito direto sobre as HUVECs, liberando ou modulando mediadores . envolvidos na hemostasia. Apesar de, a Berythractivase e a Jararagina, possuírem semelhanças estruturais, elas induzem uma dissimilar geração e liberação de moléculas endoteliais, e isso poderia explicar a diferente atividade hemorrágica existente entre elas. Baseados na regulação do gene do fator tecidual, podemos sugerir que as SVMPs estudadas agem de maneira específica, tendo a Jararagina preferencialmente uma ação local, enquanto a Berythractivase, com uma ação sobre a superfície das HUVECs, sendo uma proteína com ação pró-coagulante sistêmica.

Lonomia obliqua venom action on fibrinolytic system.

Accidental skin contact with the Lonomia caterpillar bristles causes a severe hemorrhagic syndrome. While fibrinolytic activation is considered to be the main cause of hemorrhage in Lonomia achelous envenomation, a consumptive coagulopathy was found to be a major component involved in the bleeding complications observed in patients envenomed by contact with Lonomia obliqua. Although we have previously observed that in L. obliqua envenomations, fibrinolysis activation appeared to be secondary to coagulation system activation, there are no reports regarding the ability of L. obliqua venom to activate directly fibrinolytic pathways. We examined the action of L. obliqua crude bristles extract (LOCBE) on several fibrinolytic system components. We demonstrated that LOCBE degraded the A-alpha fibrinogen chain only at high concentrations and after long incubation times. Under these conditions, LOCBE also induced prolongation of the fibrinogen clotting time, but no clot lysis was observed before 24 h. LOCBE did not contain t-PA- or u-PA-like activities. Gel filtration and SDS-PAGE showed that LOCBE did not induce FXIII digestion. In addition, no FXIII activity inhibition was detected by dansylcadaverin method. FXIII levels remained unchanged when FXIII was measured in fibrinogen-depleted LOCBE-treated rat plasma, suggesting that the observed 50% FXIII reduction in rats was related to consumption. In conclusion, our results clearly demonstrated that LOCBE did not display either FXIII inhibition or degradation nor fibrinolytic activity. Furthermore, although proteolytic activity on Aalpha fibrinogen chain was observed, cross-linked fibrin was not affected by LOCBE.