95, p ⩽ 0.01), with the notable exception of EHC-93sol, which enhanced PMA-induced response (βi-v2 = 0.024) but inhibited LPS/IFN-γ induced response (βi-v2 = −0.138). An impairment of phagocytosis in human alveolar macrophages exposed to particles has previously been shown to be independent of the type of receptors involved, whether scavenger, Nintedanib datasheet mannose, Fc or complement receptors. It was proposed that excess oxidative stress induced by particles may lead to cytoskeletal dysfunction in alveolar macrophages, impairing their motility and effector functions ( Lundborg et al., 2006). The redox-sensitive transcription factor NF-κB has been identified as

a downstream response factor common to PMA-PKC, Zymosan-Toll-like receptor 2 and LPS-Toll-like receptor 4 signal transduction pathways (Chow et al., 1999, Holden et al., 2008 and Sato et al., 2003). Toll-like receptor-mediated NF-κB activation plays an important role in the regulation of innate, as well as adaptive immune response and is a pathway evolutionarily conserved in species ranging from insects to mammals (Zhang and Ghosh, 2001), while the superoxide anion, a product of cellular respiratory burst is a trigger for PKC-mediated NF-κB activation, thus emphasizing its central role in redox-dependent pathogenesis (Ogata et al., 2000). Interestingly,

NO has been proposed as a participant in negative feedback loop regulation of particle-induced NF-κB activation in mouse macrophages (Chen et al., 1995). Our current data demonstrating a general reduction in NO production in particle-exposed and PLX3397 cost LPS/IFN-γ-stimulated cells is consistent with the participation of the NF-κB signal transduction pathway. Thus, NF-κB may represent a point of convergence in the general mechanism for the modulation by particles

of stimulant-induced respiratory burst. The results are also in agreement with our previous report of decreased NO production and iNOS protein Tacrolimus (FK506) expression in cell lines of murine monocytes exposed to urban particulate matter and subsequently stimulated with LPS/IFN-γ (Chauhan et al., 2004). A study using iNOS knockout mice indicated the involvement of iNOS in heightening the pulmonary cytokine inflammatory response to particulate matter (Becher et al., 2007). Reduction of iNOS activity may prevent cell injury by curbing excessive radical (e.g. peroxynitrite) formation. In conclusion, our data demonstrate a significant inhibitory impact of particle exposure on the respiratory burst of macrophages, revealed when the cells are challenged with a subsequent stimulant. We have extended the observations under a number of scenarios that factor-in different types of particles, soluble and insoluble fractions of particles, and different stimuli of respiratory burst that mimic the challenges to the cells during an infection.

Treatment of S2 requires that the drug crosses the blood–brain barrier (BBB); the highly specialised microvasculature that separates the cerebral tissue from the blood circulation ( Abbott et al., 2006). S1 acting drugs are pentamidine and suramin which are effective against T. b. gambiense and T. b. rhodesiense, respectively ( Brun et al., selleck chemical 2010, Sanderson et al., 2007 and Sands et al., 1985). S2 drugs are melarsoprol, eflornithine and nifurtimox. Several

recent reviews discuss the S2 acting drugs in further detail ( Brun et al., 2010 and Lutje et al., 2010). Our research group has investigated the ability of suramin, pentamidine, eflornithine and nifurtimox to cross the BBB using an in situ brain/choroid plexus perfusion technique in anaesthetised

mice ( Jeganathan et al., 2011, Sanderson et al., 2007, Sanderson et al., 2008 and Sanderson et al., 2009). Our latest study focused on nifurtimox, an anti-parasitic nitrofuran that was originally used to treat Chagas disease; a closely related condition to HAT caused by Trypanosoma cruzi ( Gonnert and Bock, 1972 and Haberkorn and Gonnert, 1972), but has since been used in compassionate treatment for HAT when other methods have failed ( Moens et al., 1984 and Van Nieuwenhove, 1992). Nifurtimox is now used against S2 in combination with eflornithine ( Checchi et al., 2007). Nifurtimox is cheap, orally active and effective against T. b. gambiense and, to a lesser extent, T. b. rhodesiense ( Bouteille et al., 2003, Haberkorn, 1979 and Lutje et al.,

2010). Importantly, our group have shown that nifurtimox Cabozantinib is able to cross the murine BBB in situ, but undergoes an efflux removal process from the brain via an unidentified process, in which the adenosine triphosphate (ATP) binding cassette (ABC) transporter P-glycoprotein, (P-gp) is not involved ( Jeganathan et al., 2011). The identify of this efflux mechanism is of special interest with the fact that nifurtimox–eflornithine combination therapy (NECT) is now becoming the first course of treatment against S2 HAT ( Yun et al., 2010), having been shown to both improve efficacy and reduce harmful side Thiamet G effects ( Priotto et al., 2007 and Priotto et al., 2009). The precise mechanisms behind the success of this particular combination therapy (CT) have yet to be fully revealed, however, it is possible CT could improve delivery to the brain. Our group have shown that nifurtimox delivery to the mouse brain is improved with the addition of the S1 acting drug pentamidine ( Jeganathan et al., 2011), which we have previously identified as being a substrate for cellular transport mechanisms at the BBB, including P-gp ( Sanderson et al., 2009). These findings highlight not only the need to elucidate the transport mechanisms utilized by nifurtimox at the BBB, but also the effect of CT on its delivery.

Phosphodiester models and mimics have been used widely to Dapagliflozin purchase understand the mechanisms

of phosphodiesterases such as nucleases and ribozymes. This section discusses examples where one or more of the bridging or non-bridging oxygen atoms associated with the phosphodiester group has been exchanged for either sulfur or fluorine. The resulting analogues are often reactive, where their altered reactivity profiles are used to probe the nature of catalysis in enzyme active sites and/or binding to metal ions therein. Some of the most poignant recent additions to the mechanistic toolbox are the phosphorothiolates (Table 2, entry 1), where a bridging oxygen atom has been replaced by sulfur. These systems have received significant attention because synthetic advances have permitted their use in oligonucleotides [14, 15 and 16]. Phosphorothiolates can also elucidate O-Mg2+ ion interactions through soft metal ion Proteasome inhibitor rescue experiments. More significantly, where a leaving

group oxygen is replaced by sulfur, the enhanced leaving group properties of thiolate anions accelerate their departure, sometimes obviating the need for catalysis, and potentially making previously kinetically silent processes rate-determining. In this vein, phosphorothiolate studies have illuminated HDV [17•] and VS [18] ribozyme systems alongside nucleobase substitutions to provide unequivocal evidence in support of general acid/base catalysis. Recent work in this area, primarily from the Piccirilli laboratory, has been reviewed [19•• and 20]. More subtle substitution of phosphodiesters can be effected through the use of isotopomeric compounds, such as 18-O

labelled species (Table 2, entry 2). Heavy atom isotope effects are challenging to determine triclocarban on a practical level, however, isotopic substitutions represent the least perturbing of all possible analogues. 5′-18O and 2′-18O isotopomeric analogues of the dinucleotide 5′-UpG-3′ were synthesised and the base-promoted cleavage kinetics of these phosphodiester systems were explored [21••]. Through these studies, the transition state for the 2′-O-transphosphorylation process was suggested to be late in nature, and solvent deuterium isotope effect studies suggest the prior formation of the 2′-alkoxide nucleophile rather than rate-determining general base catalysis by hydroxide ion. An extension of this, supplemented with computational studies, has been used to revisit the mechanism of ribonuclease A [22]. Fluorophosphonates present the possibility of concerted, diester-like transition states while offering the size and hydrogen bonding characteristics of monoesters [23]. This mixed character was used to explore the promiscuous proficiencies of phosphoryl transfer by alkaline phosphatase.

The highest DNA binding by 3-NBA in ES cells was observed at 10 μM after 24 h with 863 ± 74 adducts per 108 nucleotides (Fig. 3C). Interestingly, and in contrast to BaP, adduct levels for 3-NBA in MEFs were only 1.5-fold higher

(1266 ± 188 adduct per 108 nucleotides) under the same experimental conditions (Fig. 3D). DNA binding Dabrafenib in vivo was highest in MEFs at 10 μM after 48 h with 2478 ± 455 adducts per 108 nucleotides. Previously, in primary HUFs previously treated with 10 μM 3-NBA for 48 h, adduct levels were 680 ± 147 adducts per 108 nucleotides (Kucab et al., 2012). As 3-NBA is predominantly activated by NQO1 (Arlt et al., 2005), the expression of Nqo1 was studied in ES cells and MEFs by RT-PCR and revealed that Nqo1 mRNA expression increased in both cell types up to ∼60-fold; the induction was higher in MEFs than in ES cells ( Fig. 6C and D). This is in line with a previous study showing that Nqo1 protein levels were inducible in primary and immortal HUFs upon treatment with nitro-PAHs such as 1,8-dinitropyrene and 3-NBA ( Kucab et al., 2012). However, that study also showed that there was not a clear relationship between nitro-PAH-induced DNA adduct formation and the expression of Nqo1, suggesting

that other cytosolic nitroreductases such as xanthine oxidase might also contribute to the activation of nitro-PAHs like 3-NBA in HUFs ( Kucab et al., 2012). As shown in Fig. 5C and D, 3-NBA also induced Cyp1a1 mRNA expression, the induction in MEFs being manifoldly higher than in Sulfite dehydrogenase ES cells. Other studies have Target Selective Inhibitor Library cell line demonstrated the induction of Cyp1a1 protein levels in mouse Hepa1c1c7 cells after exposure to 3-NBA treatment ( Landvik et al., 2010) and in vivo in rats treated with 3-NBA ( Mizerovska et al., 2011, Stiborova et al., 2006 and Stiborova et al., 2008). The major activation pathway of AAI is

nitroreduction, cytosolic NQO1 being the most efficient activating enzyme while CYP1A-mediated demethylation contributes to AAI detoxification (Fig. 1C) (Stiborova et al., 2014a and Stiborova et al., 2013). Exposure to AAI resulted in loss of cell viability of both ES cells and MEFs (Fig. 2E and F). However, in contrast to 3-NBA which showed strong cytotoxicity in ES cells, AAI cytotoxicity was higher in MEFs. We therefore chose 20 μM and 50 μM AAI in MEFs while ES cells were treated with up to 100 μM for DNA adduct analysis by 32P-postlabelling (Fig. 3E and F). The AAI-induced adduct patterns in ES cells and MEFs were the same and identical to the patterns observed in kidney and ureter tissue of AAN patients (Gokmen et al., 2013 and Nortier et al., 2000). These adducts have previously been identified as 7-(deoxyadenosine-N6-yl)aristololactam I (dA-AAI; spot A1), 7-(deoxyguanosin-N2-yl)aristolactam I (dG-AAI; spot A2) and 7-(deoxyadenosin-N6-yl)aristolactam II (dA-AAII; spot A3) ( Bieler et al., 1997 and Schmeiser et al., 2014).

We observed consistent down-regulation of the bona fide YAP target gene CYR61 on YAP knockdown in all cell lines examined. CYR61 is a positive regulator of cell growth [28] and has been implicated as a proangiogenic factor in highly vascularized RCC, acting alongside vascular endothelial growth factor (VEGF) and exerting additive nonoverlapping functions [29]. CYR61 up-regulation correlated with loss of von Hippel-Lindau protein expression, although its expression was only partly dependent on Hypoxia-inducible factor 2-alpha

function, suggesting additional mechanisms that contribute to CYR61 up-regulation in RCC [29]. Furthermore, recent reports linked CYR61 with integrin-mediated cell migration and invasion Obeticholic Acid supplier in prostate cancer cell lines, click here hinting at a potential role in metastasis [30]. THBS1 is one of the most potent physiological

antiangiogenic factors and its expression has been reported as an independent prognostic factor in ccRCC with retained expression being associated with increased survival [31]. It is therefore somewhat surprising to observe down-regulation of THBS1 mRNA on YAP knockdown in all cell lines analyzed. YAP might interfere with the network of proangiogenic and antiangiogenic factors, such as CYR61 and THBS1, in ccRCC, tipping the balance toward a homeostasis that favors the proliferation and survival of the tumor cells. EDN1 and EDN2 were the most prominently downregulated genes in MZ1774 cells on YAP knockdown. Endothelins are important regulators of

kidney function, and endogenous endothelin is involved in the regulation of renal cell growth and proliferation, as well as fluid and electrolyte excretion. Production Tyrosine-protein kinase BLK of endothelins in the kidney is increased in numerous renal diseases [32], and ccRCC tumors have been reported to express EDN1 and its receptor ETA with ccRCC cell lines secreting EDN1 [33] and [34]. The selective endothelin-A receptor antagonist atrasentan has been used in combination with interferon-alpha in a phase I study in metastatic RCC, albeit with moderate clinical antitumor effects [35]. The impact of YAP knockdown on EDN2 expression was most pronounced and present in all three cell lines tested, whereas EDN1 down-regulation could be cross-validated in A498 but not in ACHN YAP knockdown cells. As ACHN YAP knockdown cells displayed the same phenotype in respect to reduced cancer cell proliferation and migration and did form smaller xenograft tumors in vivo, EDN2 seems to be one of the main effectors responsible for these effects. In line with this hypothesis, we found that YAP and EDN2 expression correlates in clinical tumor specimen of patients with ccRCC as assessed by immunohistochemistry.

Simple contrasts were applied to compare each two different positions in case of statistical significance, which was assumed for p < 0.05. Table 1 shows absolute resting values and relative maximal and stable phase (last 20 s) NVP-BGJ398 manufacturer variations to visual stimulation of HR, mean ABP, mean BFV, CVRi, CrCP, and RAP, for PCA and MCA, in supine, sitting and HUT conditions. Regarding only resting values,

repeated-measures ANOVA showed a step increase in HR from supine to HUT positions (p = 0.0001), and of mean ABP from supine to sitting (p = 0.0004), then stabilizing. There was a step decrease in mean BFV of MCA from supine to HUT conditions (p = 0.0004) but for the PCA it seemed to remain constant (p = 0.054) in all positions. Concerning resting data of cerebrovascular resistance models, RAP did not change between different positions, while CVRi and PD0325901 mouse CrCP resting values progressively increased from supine to HUT conditions, in both MCA (p = 0.00001 and p = 0.0002, respectively) and PCA (p = 0.0002 and p = 0.00005, respectively), although not reaching statistical significance between sitting and HUT in the case of CVRi

of PCA (p = 0.053). The variation of the parameters with visual stimulation can be visualized in Fig. 1A–F. Mean BFV in the PCA, had similar responses to visual stimulation in all positions (Fig.

1A, maximal p = 0.076; stable phase p = 0.176). All cerebrovascular resistance parameters decreased with visual stimulation in the three positions, but showed different patterns in response to orthostatic challenge: variation of CrCP diminished progressively between supine and HUT (maximal and stable phase p = 0.001); CVRi decreased slightly but significantly more from sitting Thalidomide to HUT positions (maximal p = 0.036; stable phase p = 0.033). RAP seemed to have decreased more in HUT conditions but there was no statistical significance (maximal p = 0.077; stable phase p = 0.188). Although the MCA territory was used as a control, being theoretically a non-stimulated territory, it registered, similarly across all conditions, a small amplitude increment in mean BFV (5–10%), as well as a decrement of CVRi (6–9%), RAP (9–11%) and CrCP (11–17%) at maximal evoked flow phase, which then tended to decrease in the stable phase. For the MCA significant changes were only observed for BFV in maximal (p = 0.035) and CVRi in maximal (p = 0.029) and stable phases (p = 0.043). Regarding systemic hemodynamic data, the changes of ABP and HR with stimulation ranged no more than 4%, with no significant differences between positions, except for maximal increment of ABP which was inferior during HUT compared to supine condition (p = 0.045).

The absorbance was measured at 532 nm and results were expressed as MDA equivalents formed by Fe2+ and

H2O2. Nitric oxide was generated from spontaneous decomposition of sodium nitroprusside in 20 mM phosphate buffer (pH 7.4). Once generated NO interacts with oxygen to produce nitrite ions, which were measured by the Griess reaction (Basu and Hazra, 2006). The reaction mixture (1 ml) containing 10 mM sodium nitroprusside (SNP) in phosphate buffer and ATR at different concentrations were incubated at 37 °C for 1 h. A 0.5 ml aliquot was taken and homogenized with 0.5 ml Griess reagent. The absorbance of chromophore was measured at 540 nm. Percent inhibition of nitric oxide generated was measured by comparing the absorbance values of negative controls (only 10 mM sodium nitroprusside and vehicle) find more and assay preparations. Results were expressed as percentage selleck chemical of nitrite formed by ATR alone. The ability

of ATR to scavenge H2O2 (“catalase-like activity” or “CAT-like activity”) was measured as described previously (Aebi, 1984). Briefly, H2O2 diluted in 0.02 M phosphate buffer (pH 7.0) to obtain a 5 mM final concentration was added to microplate wells in which different concentrations was placed. The microplate was immediately placed to monitor the rate of H2O2 decomposition in the microplate reader set at 240 nm. The ability of ATR to scavenge superoxide anion (“superoxide dismutase-like activity” or “SOD-like activity”) was measured as previously described. ATR was mixed to native purified catalase (100 U/ml stock solution) in glycine buffer (50 mM, pH 10.2). Superoxide generation was initiated by addition of adrenaline 2 mM and adrenochrome formation was monitored at 480 nm for 5 min at 32 °C. Superoxide production was determined by monitoring the reaction curves of samples and measured as percentage of the rate of adrenaline auto-oxidation into adrenochrome (Bannister and Calabrese, 1987). SH-SY5Y cells were cultured in 10% FBS DMEM/F12 medium. Cells were used for cytotoxicity measurements when reached 70–90% confluence. Cells were treated with different concentrations

of ATR alone or in the presence of H2O2 400 μM for 3 h, and cell viability Montelukast Sodium was assessed by the MTT assay. This method is based on the ability of viable cells to reduce MTT (3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide) and form a blue formazan product. MTT solution (sterile stock solution of 5 mg/ml) was added to the incubation medium in the wells at a final concentration of 0.2 mg/ml. The cells were left for 45 min at 37° C in a humidified 5% CO2 atmosphere. The medium was then removed and plates were shaken with DMSO for 30 min. The optical density of each well was measured at 550 nm (test) and 690 nm (reference). Data are expressed as mean ± SEM. The obtained data was evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s test. All tests were performed in triplicate. Data analyses were performed using the GraphPad Prism 5.

Furthermore, we found a relationship between physical activity and fallers: residents who walk occasionally or frequently are significantly more often

a faller. Overall, malnourished LTC residents are in general: (1) more prone to be a faller and (2) less active. However, we also observed that the rate of fallers is higher among the relatively active LTC residents. As these observations seem contradictory, we also investigated the role of activity in the relationship between nutritional status and fallers. From this analysis it appeared that in both the active and the inactive group, malnutrition is related to fallers in LTC settings. When specifically looking at the residents who walked occasionally or frequently, the fall rate was higher in those who walked occasionally and highest when concomitantly also malnourished. A plausible explanation for see more the observations with regard

to activity may be that the activity-item of the Braden scale provides rather a rough classification of (in)activity and lacks sensitivity in detecting small differences in the activity level of LTC residents. It is also not surprising that increased activity, as seen in the categories occasionally walking and frequently walking, increases the occasions where a fall can occur compared with the categories bedfast and chairfast (Bueno-Cavanillas et al., 2000, Graafmans et al., 1996, Halfens this website et al., 2007, Halfens et al., 2008, Halfens et al., 2010, Halfens et al., 2009 and Kiely et al., 1998). Therefore, further research is warranted

on this issue and measurement of actual physical activity is preferred. Finally, the influence of nutritional intervention on the relation between nutritional status and fallers was investigated. Specifically in malnourished residents, the results suggest a positive effect of nutritional intervention on the risk of being a faller. This strengthens the observed relationship between nutritional status and fallers. However, future prospective research is essential to further substantiate this finding among LTC residents PLEKHM2 and to gain a better understanding of the potential beneficial role of nutritional intervention and specific nutritional components in falls prevention. At present, few data regarding fall-related nutritional intervention are available, and only vitamin D supplementation has been shown effective in reducing the rate of falls in nursing care facilities (Cameron et al., 2010). There are also some limitations of the present study that need to be mentioned. First, a particular difficulty with cross-sectional studies focusing on relationships is the fact that causality cannot be determined, which can be addressed in future intervention studies.

The numbers of children that presented detectable IgA antibodies to antigens of each Streptococcal species and mean numbers of reactive bands detected are shown in Table 1. Although IgA antibody responses were detected more frequently to S. mitis Ags (n = 23,

[11 PT and 12 FT]) when compared to S. mutans antigens buy Idelalisib (n = 18, [7 PT and 11 FT]) those differences were not significant (Mann–Whitney, P > 0.05). Additionally, the number of IgA-reactive bands to S. mitis antigens was significantly higher in FT than in PT children (Mann–Whitney U test, P ≤ 0.05). Six percent of the SDS–PAGE gels analysed allowed the visualization of important antigens from S. mutans: Ag I/II (185 kDa), GTF C (160 kDa) and GbpB (56 kDa) and of S. mitis: IgA-protease (202 kDa). Twenty-one percent of children (n = 10, [3PT and 7FT]) had IgA reactive to Ag 202 kDa–S. mitis and 16.5 (n = 8, [2PT and 6FT]) and 17 (n = 8 [4FT and 4 PT]) % of children presented IgA reactive

to 185 and 160 kDa–S. mutans Ags respectively ( Table 1). We did not find children with IgA reactive with bands in the 56 kDa region of S. mutans blots. There Nutlin 3a were no significant differences in the number of PT and FT children with IgA responses to these antigens (Qui Square test, q < 2.01; P > 0.27). There were variations in the intensities and numbers of IgA antibody reactions with the recognized bands amongst children in both groups. Table 1 shows the sums of intensities of IgA reactions with all bands detected for each species (total intensities) observed in children of the FT and PT groups. In general, FT children presented the highest intensity of IgA to all antigens tested but

those differences were not statistically significant (Mann–Whitney U test, P > 0.2), likely due to the high variability in intensities of response amongst children of each group. The results showed that SIgA antibody from 10 samples (3 PT and 7 FT) tested did not react with E. faecalis antigens, as SIgA responses to S. mutans and S. mitis were not reduced by E. faecalis cross-adsorption. On the other hand, when samples (n = 10) were adsorbed with cells of S. mitis, there were mean reductions of 22% of SIgA to S. mutans in 5 children (4 FT and 1 PT). In the same children (n = 5), there was also a mean reduction of 45% of SIgA to S. mitis when samples were adsorbed previously L-NAME HCl with cells of S. mutans. Salivary IgA antibodies play several roles in the modulation of the establishment of the microbiota compatible with health homeostasis19 and form a first line of defence against specific pathogens.19 Salivary IgA antibodies neutralize antigenic components involved in microbial virulence and might block surface adhesins important for colonization of the mucosa.20 In the saliva, secretory IgA predominates, but early in life, IgM is also normally detected.6 Previously, it was described that IgA can be detected in saliva at birth.

plumieri venom on washed rabbit erythrocytes ( Andrich et al., 2010). As previously described for other hemolytic factors purified from stonefish venoms, such as stonustoxin (SNTX), trachynilysin (TLY) and neoverrucotoxin (neoVTX) ( Poh et al., 1991, Colasante et al., 1996 and Ueda et al., 2006), Sp-CTx

elicits other pharmacological activities. Andrich et al., 2010, have demonstrated that Sp-CTx causes a biphasic response on phenylephrine pre-contracted rat aortic ring, characterized by an endothelium and dose-dependent relaxation phase followed by a contractile phase. The estimation of Sp-CTx native selleck chemicals llc molecular mass was performed by size exclusion chromatography and demonstrated that it is a 121 kDa protein. Further physicochemical studies revealed its glycoprotein nature and suggested a dimeric constitution, comprising subunits of approximately 65 kDa (Andrich et al., 2010). However, there is very little information concerning the mechanism involved in the Sp-CTx hemolytic activity. Essentially, this is due to the extreme lability of fish venom toxins, since most of their biological properties are lost during storage. Their instability has made it difficult to study piscine venoms, and this may be explained by the easily denatured high-molecular-mass proteins and also by the presence of proteolytic enzymes in these venoms (Perriere et al., 1988, Garnier

et al., 1995 and Abe et al., 1996). Thus, at the present work we aimed to elucidate the mechanisms involved in the hemolytic Epacadostat purchase activity induced by Sp-CTx and to determine some biochemical properties of this toxin. Specimens learn more of S. plumieri (10–26 cm in length) were collected in shallow seashore in the state of Espírito Santo, Brazil, and kept alive in oxygenated seawater aquarium. The captures were authorized by the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis – IBAMA (the Brazilian Public Agency for Environment Affairs). The venom from fin spines was extracted according to the batch method previously described by Schaeffer et al. (1971) with few modifications.

The entire extraction process was carried out at 4 °C. Freshly extracted soluble crude venom was immediately used for purification procedure and hemolytic assay. The protein concentration was determined by the Lowry method ( Lowry et al., 1951) using bovine serum albumin as standard. Sp-CTx was purified from the crude venom by salt precipitation followed by two chromatographic steps and the presence of protein in the chromatographic fractions was monitored by absorbance at 218 nm. Cytolytic fractions were identified by hemolytic assay on erythrocytes as described in item 2.3. Venom aqueous solution containing 48.7 mg of protein was submitted to two steps ammonium sulfate precipitation at 4 °C, beginning at 15% up to 35%. Precipitate of each step was collected by centrifugation (30,000 × g/30 min) and dissolved in 2 mL of 20 mM sodium phosphate buffer (PB) containing 0.15 M NaCl, pH 7.4 (PBS).