Aggregation susceptibility on phototransformation of hematoporphyrin derivatives

Photosensitizers used in PDT suffer degradation by light. In this work, photobleaching of Photogem((R)) (PG), Photofrin((R)) (PF), and Photosan((R)) (PS), hematoporphyrin derivatives, were induced by light in the presence or absence of 1% Triton X-100. The degradation efficiency in the absence of 1% Triton X-100 follows the sequence: Pf > PF > Ps, which means that PF presented a greater degradation than PF and PS. Forever, in the presence of the surfactant the degradation efficiency is different: PF congruent to PS > PF. Besides aggregation susceptibility, studies in cell culture (tumor and non tumor cells) and in animals (depth of necrosis) were performed, trying to correlate the stability of these photosensitizers with their photodynamic effect. The results suggest that PF presents higher light induced photo-cytotoxicity than PF and PS for both types of cells. For the depth of necrosis studies, more aggregated photosensitizer showed a longer time to accumulate in liver (30 min for PG, 120 h for PF and 720 h for PS). The, to establish an ideal dosimetry in PDT, one must consider the intrinsic physical chemistry characteristics of the photosensitizer as well as their ability to undergo photobleaching.

Experimental evidence and model explanation for cell population characteristics modification when applying sequential photodynamic therapy

We present experimental evidence of the existence of cell variability in terms of threshold light dose for Hep G2 (liver cancer cells) cultured. Using a theoretical model to describe the effects caused by successive photodynamic therapy (PDT) sessions, and based on the consequences of a partial response we introduce the threshold dose distribution concept within a tumor. The experimental model consists in a stack of flasks, and simulates subsequent layers of a tissue exposed to PDT application. The result indicates that cells from the same culture could respond in different ways to similar PDT induced-damages. Moreover, the consequence is a partial killing of the cells submitted to PDT, and the death fraction decreased at each in vitro PDT session. To demonstrate the occurrence of cell population modification as a response to PDT, we constructed a simple theoretical model and assumed that the threshold dose distribution for a cell population of a tumor is represented by a modified Gaussian distribution.

trans, trans-2,4-decadienal induces mitochondrial dysfunction and oxidative stress

Lipid peroxidation produces a large number of reactive aldehydes as secondary products. We have previously shown that the reaction of cytochrome c with trans,trans-2, 4-decadienal (DDE), an aldehyde generated as a product of lipid peroxidation in cell membranes, results in the formation of adducts. Mass spectrometry analysis indicated that His-33, Lys-39, Lys-72 and Lys-100 in cytochrome c were modified by DDE. In the present work, we investigated the effect of DDE on isolated rat liver mitochondria. DDE (162 mu M) treatment increases the rate of mitochondrial oxygen consumption. Extensive mitochondrial swelling upon treatment with DDE (900 nM-162 mu M) was observed by light scattering and transmission electron microscopy experiments. DDE-induced loss of inner mitochondrial membrane potentials, monitored by safranin O fluorescence, was also observed. Furthermore, DDE-treated mitochondria showed an increase in lipid peroxidation, as monitored by MDA formation. These results suggest that reactive aldehydes promote mitochondrial dysfunction.

Mitochondrial energy metabolism in neurodegeneration associated with methylmalonic acidemia

Methylmalonic acidemia is one of the most prevalent inherited metabolic disorders involving neurological deficits. In vitro experiments, animal model studies and tissue analyses from human patients suggest extensive impairment of mitochondrial energy metabolism in this disease. This review summarizes changes in mitochondrial energy metabolism occurring in methylmalonic acidemia, focusing mainly on the effects of accumulated methylmalonic acid, and gives an overview of the results found in different experimental models. Overall, experiments to date suggest that mitochondrial impairment in this disease occurs through a combination of the inhibition of specific enzymes and transporters, limitation in the availability of substrates for mitochondrial metabolic pathways and oxidative damage.

Antioxidant effect of functionalized alkyl-organotellurides: a study in vitro

We evaluated the in vitro antioxidant effect of alkyl-organotellurides A-D on lipid peroxidation and protein carbonylation in rat liver homogenates. The thiol oxidase and thiol peroxidase-like activities of compounds were investigated. delta-Aminolevulinic acid dehydratase (delta-ALA-D) activity was determined in rat liver homogenates. Compounds A-D protected against lipid peroxidation induced by Fe(2+)/EDTA and sodium nitroprusside (SNP). According to the confidence limits of the IC(50) values of compounds A-D, the IC(50) values for organotellurides followed the order: C (0.30 mu M) <= B (0.40 mu M) < D (0.68 mu M) < A (2.90 mu M), for Fe(2+)/EDTA, and B (0.21 mu M) <= C (0.33 mu M) < D (0.43 mu M) < A (1.21 mu M) for SNP-induced lipid peroxidation. Compounds A-D reduced protein carbonyl content to control levels. The results demonstrated an inverse correlation between thiol oxidase and delta-ALA-D activities. This study supports an antioxidant effect of organotellurides A-D on rat liver.

Methylmercury localization in Danio rerio retina after trophic and subchronic exposure: A basis for neurotoxicology

Methylmercury is a known neurotoxic organometal which affects visual functions and few studies concerns to wild fish are available. The autometallography mercury distribution in the retina of Danio rerio was mapped using light and electron microscopy. Abundant mercury deposits were found in the photoreceptor layer (outer and inner segments of the photoreceptors) and in the inner and outer nuclear layers. Occasionally, the presence of mercury deposits in plexiform layers was observed and very rarely in the ganglion cell layer. Also the occurrence of mercury deposits in cells from the disc region was observed, but not in the nerve fiber layer. An interesting difference was found between mercury accumulation in the central and peripheral regions of the retina. These results demonstrate that mercury after trophic exposure to Danio rerio is able to cross the blood-retina barrier and accumulate in the cells of the retina even under subchronic exposure. (C) 2010 Elsevier Inc. All rights reserved.

A mechanistic study on altered pharmacokinetics of irinotecan by St. Johns wort

Irinotecan (CPT-11) is an important anticancer drug in management of advanced colon cancer. A marked protective effect on CPT-11-induced blood and gastrointestinal toxicity is obtained by combination of St. John's wort (SJW) in recent clinical and rat studies. However, the mechanism is unclear. This study aimed to explore the effects of SJW on the pharmacokinetics of CPT-11 and its major metabolites (SN-38 and SN-38 glucuronide) in rats and the underlying mechanisms using several in vitro models. Short-term (3 days) and long-term (14 days) pretreatment with SJW were conducted in rats to examine the effects of co-administered SJW on the plasma pharmacokinetics of CPT-11, SN-38 and SN- 38 glucuronide. Rat liver microsomes and a rat hepatoma cell line, H4-II-E cells, were utilized to study the effects of aqueous and ethanolic extracts (AE and EE) and major active components (hyperforin, hypericin and quercetin) of SJW on CPT-11 and SN-38 metabolism and intracellular accumulation. Co-administered SJW for consecutive 14 days significantly decreased the initial plasma concentration (C0) of CPT-11, the area under the concentration-time curve (AUC0-10hr) and maximum plasma concentration (Cmax) of SN-38. The ethanolic extracts (EE) of SJW at 5 μ g/ml significantly decreased SN-38 glucuronidation by 45% (P < 0.05) in rat hepatic microsomes. Pre-incubation of aqueous SJW extracts (AE) at 10 g/ml, SJW EE at 5 μg/ml, and quercetin at 10μ M significantly increased the glucuronidation of SN-38 in H4- II-E cells. A 2-hr pre-incubation of quercetin (100μ M) significantly increased the intracellular accumulation of CPT-11 (P < 0.05). However, pre-incubation of hypericin (20 nM and 200 nM) and hyperforin (1μ M) significantly decreased the intracellular accumulation of CPT-11. In addition, pre-incubation of hypericin, SJW EE and quercetin significantly increased the intracellular accumulation of SN-38. Aqueous and ethanolic SJW extracts and its major active components did not alter the plasma protein binding of CPT-11 and SN-38. These results indicated that the aqueous and ethanolic extracts of SJW and its major active components could markedly alter glucuronidation of SN-38 and intracellular accumulation of CPT-11 and SN-38, which probably provides partial explanation for the altered plasma pharmacokinetics of CPT-11 and SN-38 and the antagonizing effects on the toxicities of CPT-11. Further studies are needed to explore the role of both pharmacokinetic and pharmacodynamic components in the protective effect of SJW against the toxicities of CPT-11.

Cytochrome bc1 regulates the mitochondrial permeability transition by two distinct pathways

The mitochondrial permeability transition (MPT) pore is a calcium-sensitive channel in the mitochondrial inner membrane that plays a crucial role in cell death. Here we show that cytochrome bc₁ regulates the MPT in isolated rat liver mitochondria and in CEM and HL60 cells by two independent pathways. Glutathione depletion activated the MPT via increased production of reactive oxygen species (ROS) generated by cytochrome bc₁. The ROS producing mechanism in cytochrome bc₁ involves movement of the "Rieske" iron-sulfur protein subunit of the enzyme complex, because inhibition of cytochrome bc₁ by pharmacologically blocking iron-sulfur protein movement completely abolished ROS production, MPT activation, and cell death. The classical inhibitor of the MPT, cyclosporine A, had no protective effect against MPT activation. In contrast, the calcium-activated, cyclosporine A-regulated MPT in rat liver mitochondria was also blocked with inhibitors of cytochrome bc₁. These results indicate that electron flux through cytochrome bc1 regulates two distinct pathways to the MPT, one unregulated and involving mitochondrial ROS and the other regulated and activated by calcium.

A mechanistic study on reduced toxicity of irontecan by coadministered thalidomide, a tumor necrosis factor-a inhibitor

Dose-limiting diarrhea and myelosuppression compromise the success of irinotecan (7-ethyl-10-[4-[1-piperidino]-1-piperidino] carbonyloxycamptothecin) (CPT-11)-based chemotherapy. A recent pilot study indicates that thalidomide attenuates the toxicity of CPT-11 in cancer patients. This study aimed to investigate whether coadministered thalidomide modulated the toxicities of CPT-11 and the underlying mechanisms using several in vivo and in vitro models. Diarrhea, intestinal lesions, cytokine expression, and intestinal epithelial apoptosis were
monitored. Coadministered thalidomide (100 mg/kg i.p. for 8 days) significantly attenuated body weight loss, myelosuppression, diarrhea, and intestinal histological lesions caused by CPT-11 (60 mg/kg i.v. for 4 days). This was accompanied by inhibition of tumor necrosis factor-, interleukins 1 and 6 and interferon-, and intestinal epithelial apoptosis. Coadministered
thalidomide also significantly increased the systemic exposure of CPT-11 but decreased that of SN-38 (7-ethyl-10-hydroxycampothecin). It significantly reduced the biliary excretion and cecal exposure of CPT-11, SN-38, and SN-38 glucuronide. Thalidomide hydrolytic products inhibited hydrolysis of CPT-11 in rat liver microsomes but not in primary rat hepatocytes. In addition, thalidomide and its major hydrolytic products, such as phthaloyl glutamic acid (PGA), increased the intracellular accumulation of CPT-11 and SN-38 in primary rat hepatocytes. They also significantly decreased the transport of CPT-11 and SN-38 in Caco-2 and parental MDCKII cells. Thalidomide and PGA also significantly inhibited P-glycoprotein (PgP/MDR1), multidrug resistance-associated protein (MRP1)- and MRP2-mediated CPT-11 and SN-38 transport in MDCKII cells. These results provide insights into the pharmacodynamic and  pharmacokinetic mechanisms for the protective effects of thalidomide against CPT-11-induced intestinal toxicity.

A mechanistic study of the intestinal absorption of crypotanshinone, the major active constituent of Saliva miltiorrhiza

The nature of intestinal absorption of most herbal medicine is unknown. Cryptotanshinone (CTS) is the principal active constituent of the widely used cardiovascular herb Salvia miltiorrhiza (Danshen). We investigated the oral bioavailability of CTS in rats and the mechanism for its intestinal absorption using several in vitro and in vivo models:1) Caco-2 cell monolayers; 2) monolayers of MDCKII cells overexpressing P-glycoprotein
(PgP); and 3) single-pass rat intestinal perfusion with mesenteric vein cannulation. The systemic bioavailabilities of CTS after oral and intraperitoneal administration at 100 mg/kg were 2.05 and 10.60%, respectively. In the perfused rat intestinal model, permeability coefficients based on CTS disappearance from the luminal perfusate (P_lumen) were 6.7- to 10.3-fold higher than permeability coefficients based on drug appearance in venous blood (P_blood). P_blood significantly increased in the presence of the P-gP inhibitor, verapamil. CTS transport across Caco-2 monolayers was pH-, temperature- and ATP-dependent. The transport from the apical (AP) to the basolateral (BL) side was 3- to 9-fold lower than that from the BL to the AP side. Inclusion of verapamil (50 µM) in both AP and BL sides abolished the polarized CTS transport across Caco-2 cells. Moreover, CTS was significantly more permeable in the BL to AP than in the AP to BL direction in MDCKII and MDR1-MDCKII cells. The permeability coefficients in the BL to AP direction were significantly higher in MDCKII cells overexpressing PgP. These findings indicate that CTS is a substrate for PgP that can pump CTS into the luminal side.

Pharmacokinetic mechanisms for reduced toxicity of irinotecan by coadministered Thalidomide

The clinical use of irinotecan (CPT-11) is hindered by dose-limiting diarrhea and myelosuppression. Recent clinical studies indicate that thalidomide, a known tumor necrosis factor-alpha inhibitor, ameliorated the toxicities induced by CPT-11. However, the mechanisms for this are unknown. This study aimed to investigate whether combination of thalidomide modulated the toxicities of CPT-11 using a rat model and the possible role of the altered pharmacokinetic component in the toxicity modulation using in vitro models. The toxicity model was constructed by treatment of healthy rats with CPT-11 at 60 mg/kg per day by intravenous (i.v.) injection. Body weight, acute and delayed-onset diarrhea, blood cell counts, and macroscopic and microscopic intestinal damages were monitored in rats treated with CPT-11 alone or combined therapy with thalidomide at 100 mg/kg administered by intraperitoneal (i.p.) injection. Single dose and 5-day multiple-dose studies were conducted in rats to examine the effects of concomitant thalidomide on the plasma pharmacokinetics of CPT-11 and its major metabolites SN-38 and SN-38 glucuronide (SN-38G). The effect of CPT-11 on thalidomide's pharmacokinetics was also checked. Rat liver microsomes and a rat hepatoma cell line, H4-II-E cells, were used to study the in vitro metabolic interactions between these two drugs. H4-II-E cells were also used to investigate the effect of thalidomide and its hydrolytic products on the transport of CPT-11 and SN-38. In addition, the effect of thalidomide and its hydrolytic products on rat plasma protein binding of CPT-11 and SN-38 was examined. Administration of CPT-11 by i.v. for 4 consecutive days to rats induced significant body weight loss, decrease in neutrophil and lymphocyte counts, severe acute- and delayed-onset diarrhea, and intestinal damages. These toxicities were alleviated when CPT-11 was combined with thalidomide. In both single-dose and 5-day multiple-dose pharmacokinetic study, coadministered thalidomide significantly increased the area under the plasma concentration-time curve (AUC) of CPT-11, but the AUC and elimination half-life (t(1/2)) of SN-38 were significantly decreased. However, CPT-11 did not significantly alter the pharmacokinetics of thalidomide. Thalidomide at 25 and 250 microM and its hydrolytic products at a total concentration of 10 microM had no significant effect on the plasma protein binding of CPT-11 and SN-38, except for that thalidomide at 250 microM caused a significant increase in the unbound fraction (f(u)) of CPT-11 by 6.7% (P < 0.05). The hydrolytic products of thalidomide (total concentration of 10 microM), but not thalidomide, significantly decreased CPT-11 hydrolysis by 16% in rat liver microsomes (P < 0.01). The formation of both SN-38 and SN-38G from CPT-11, SN-38 glucuronidation, or intracellular accumulation of both CPT-11 and SN-38 in H4-II-E cells followed Michaelis-Menten kinetics with the one-binding site model being the best fit for the kinetic data. Coincubation or 2-hr preincubation of thalidomide at 25 microM and 250 microM and its hydrolytic products at 10 microM did not show any significant effects on CPT-11 hydrolysis and SN-38 glucuronidation. However, preincubation of H4-II-E cells with thalidomide (250 microM), its hydrolytic products (total concentration of 10 microM), or phthaloyl glutamic acid (one major thalidomide hydrolytic product, 10 microM) significantly increased the intracellular accumulation of SN-38, but not CPT-11 (P < 0.01). The dose-limiting toxicities of CPT-11 were alleviated by combination with thalidomide in rats and the pharmacokinetic modulation by thalidomide may partially explain its antagonizing effects on the toxicities of CPT-11. The hydrolytic products of thalidomide, instead of the parental drug, modulated the hepatic hydrolysis of CPT-11 and intracellular accumulation of SN-38, probably contributing to the altered plasma pharmacokinetics of CPT-11 and SN-38. Further studies are needed to explore the role of both pharmacokinetics and pharmacodynamic components in the protective effect of thalidomide against the toxicities of CPT-11.

Induction of propranolol metabolism by Ginkgo biloba extract EGb 761 in rats

Ginkgo biloba is one of the most popular herbal medicines in the world, due to its purported pharmacological effects, including memory-enhancing, cognition-improving, and antiplatelet effects. When used in the elderly, Ginkgo has a high potential for interactions with cardiovascular drugs. This study aimed to investigate the effects of the standard Ginkgo biloba extract (EGB 761) treatment on the pharmacokinetics of propranolol and its metabolism to form Ndesisopropylpropranolol (NDP) in rats. We also examined the activity and expression of cytochrome P450 (CYP) 1A and other CYPs in rats treated with EGb 761 at 10 and 100 mg/kg/day for 10 days. A single oral dose of propranolol (10 mg/kg) was administered on day 11 and the concentrations of both propranolol and NDP were determined using validated liquid chromatography-mass spectrometry (LC-MS) methods. The levels of mRNA and protein of various CYPs were determined by RT-PCR and Western blotting analysis, respectively. Pretreatment of EGb 761 at 100 mg/kg, but not 10 mg/kg, for 10 days significantly reduced the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (C max) of propranolol, whereas those values of NDP were significantly increased. CYP1A1, 1A2, 2B1/2, and 3A1 activities and gene expression in the rat liver were significantly increased in a dose-dependent manner by pretreatment with EGb 761. The ex-vivo formation of NDP in liver microsomes from rats pretreated with EGb 761 was markedly enhanced. The formation of NDP from propranolol in liver microsomes was significantly inhibited by α- naphthoflavone (ANF, a selective CYP1A2 inhibitor), but not by quinidine (a CYP2D inhibitor). These results indicated that EGb 761 pretreatment decreased the plasma concentrations of propranolol by accelerated conversion of parental drug to NDP due to induction of CYP1A2. EGb 761 pretreatment also significantly induced CYP2B1/2 and CYP3A1, suggesting potential interactions with substrate drugs for these two enzymes. Further study is needed to explore the potential for gingko-drug interactions and the clinical impact.

Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

This paper describes the development of a microfluidic methodology, using RNA extraction and reverse transcription PCR, for investigating expression levels of cytochrome P450 genes. Cytochrome P450 enzymes are involved in the metabolism of xenobiotics, including many commonly prescribed drugs, therefore information on their expression is useful in both pharmaceutical and clinical settings. RNA extraction, from rat liver tissue or primary rat hepatocytes, was performed using a silica-based solid-phase extraction technique. Following elution of the purified RNA, amplification of target sequences for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the cytochrome P450 gene CYP1A2, was carried out using a one-step reverse transcription PCR. Once the microfluidic methodology had been optimized, analysis of control and 3-methylcholanthrene-induced primary rat hepatocytes were used to evaluate the system. As expected, GAPDH was consistently expressed, whereas CYP1A2 levels were found to be raised in the drug-treated samples. The proposed system offers an initial platform for development of both rapid throughput analyzers for pharmaceutical drug screening and point-of-care diagnostic tests to aid provision of drug regimens, which can be tailor-made to the individual patient.

Dietary micronutrients and in vivo n-3 LC-PUFA biosynthesis in Atlantic salmon

Aquaculture, and in particular Atlantic salmon culture, is expected to deliver n. -3 long-chain polyunsaturated fatty acid (n. -3 LC-PUFA) rich products. Nevertheless, the availability of n. -3 LC-PUFA rich raw materials for aquafeed is dwindling, and at an ever increasing market price. Thus, there is the need to better understand the in vivo n. -3 LC-PUFA biosynthetic capabilities of cultured fish to enable the possible maximization of dietary 18:3n. -3 (ALA) bioconversion to 20:5n. -3 (EPA) and 22:6n. -3 (DHA). The cofactors and coenzymes involved in this metabolic pathway have so far received limited research attention. In this study, juvenile Atlantic salmon were fed an ALA-rich diet with no, normal, or over-fortified inclusion of those micronutrients reported to be essential cofactors (iron; zinc; magnesium) and coenzymes (riboflavin; biotin; niacin) for the fatty acid elongase and desaturase enzymes. The results showed that reduced dietary inclusion of these micronutrients impaired the normal n. -3 LC-PUFA biosynthetic capabilities of fish, whereas the over fortification did not provide any additional benefit. This study provides new knowledge on micronutrients and lipid metabolism interactions in a commercially important cultured species, and is envisaged to be a useful contribution towards developing more sustainable and commercially viable aquafeed for the future.Statement of relevance. This work is the continuation and extension of a previous study (Lewis et al., 2013, Aquaculture 412/413, 215-222) in which we explored the physiological roles and potential effects of micronutrients on fatty acid metabolism in cultured fish. The present study differed from the previous in the blend of minerals and vitamins used, the species, the fatty acid composition of the test diet, and the inclusion also of a negative control. The results are most interesting, showing that riboflavin (B2), biotin (B7), and niacin (B3), Iron (Fe), Magnesium (Mg) and Zinc (Zn) are all required for proper fatty acid bioconversion, but also that a dietary over-fortification does not translate into proportional improved bioconversion.

Renal and antibacterial effects induced by myotoxin I and II isolated from Bothrops jararacussu venom

Bothrops jararacussu myotoxin I (BthTx-I; Lys 49) and II (BthTX-II; Asp 49) were purified by ion-exchange chromatography and reverse phase HPLC. In this work we used the isolated perfused rat kidney method to evaluate the renal effects of B. jararacussu myotoxins I (Lys49 PLA(2)) and II (Asp49 PLA(2)) and their possible blockage by indomethacin. BthTX-1 (5 mu g/ml) and BthTX-II (5 mu g/ml) increased perfusion pressure (PP; ct(120) = 110.28+/-3.70 mmHg; BthTX I = 171.28+/-6.30* mmHg; BthTX II = 175.50+/-7.20* mmHg), renal vascular resistance (RVR; ct(120) = 5.49+/-0.54 mmHg/ml.g(-1) min(-1); BthTX I = 8.62+/-0.37* mmHg/ml g(-1) min(-1); BthTX II=8.9+/-0.36* mmHg/ml g(-1) min(-1)), urinary flow (UF; ct(120)= 0.14+/-0.01 ml g(-1) min(-1); BthTX I=0.32+/-0.05* ml g(-1) min(-1); BthTX II=0.37+/-0.01* ml g(-1) min(-1)) and glomerular filtration rate (GFR; ct(120)=0.72+/-0.10 ml g(-1) min(-1); BthTX I=0.85+/-0.13* ml g(-1) min(-1); BthTX II=1.22+/-0.28* ml g(-1) min(-1)). In contrast decreased the percent of sodium tubular transport (%TNa+; ct(120)=79,76+/-0.56; BthTX I=62.23+/-4.12*; BthTX II=70.96+/-2.93*) and percent of potassium tubular transport (%TK+;ct(120)=66.80+/-3.69; BthTX I=55.76+/-5.57*; BthTX II=50.86+/-6.16*). Indomethacin antagonized the vascular, glomerular and tubular effects promoted by BthTX I and it's partially blocked the effects of BthTX II. In this work also evaluated the antibacterial effects of BthTx-I and BthTx-II against Xanthomonas axonopodis. pv. passiflorae (Gram-negative bacteria) and we observed that both PLA2 showed antibacterial activity. Also we observed that proteins Also we observed that proteins chemically modified with 4-bromophenacyl bromide (rho-BPB) decrease significantly the antibacterial effect of both PLA(2). In conclusion, BthTx I and BthTX II caused renal alteration and presented activity antimicrobial. The indomethacin was able to antagonize totally the renal effects induced by BthTx I and partially the effects promoted by BthTx II, suggesting involvement of inflammatory mediators in the renal effects caused by myotoxins. In the other hand, other effects could be independently of the enzymatic activity of the BthTX II and the C-terminal domain could be involved in both effects promoted for PLA(2). (C) 2005 Elsevier Ltd. All rights reserved.