6, 200 mM NaCl, 100 mM CaCl2, and 1% Triton X-100). After Selleckchem BMS-734016 centrifugation (12,000 × g, 10 °C, 10 min), protein concentration in supernatant aliquots was determined ( Lowry et al., 1951), and equal amounts of total protein loaded for zymography (60 μg/lane) to determine gelatinase activity ( Heussen and Dowdle, 1980). Zymogram gels consisted of 7.5% polyacrylamide-SDS impregnated with 2 mg/ml type A gelatin from porcine skin (Sigma, St. Louis, MI) and 4% polyacrylamide-SDS for stacking gels. Gels were further washed twice for 30 min in 2.5% Triton X-100 solution, then incubated at 37 °C for 24 h in substrate buffer (10 mM Tris–HCl buffer, pH 7.5, with 5 mM CaCl2, 1 mM ZnCl2). Gels were stained with 30%

methanol/10% acetic acid solution containing 0.5% brilliant blue R-250 (Sigma) and discolored with the same

solution without Alectinib mw dye. Quantitative image analysis was performed with software Scion Image for Windows (Scion Corporation, National Institutes of Health; Bethesda, MD). Statistical analysis was carried out using GraphPad Prism software (GraphPad Software Inc., San Diego, CA) with one-way analysis of variance (ANOVA), Tukey’s multiple comparisons test and unpaired Student’s t-test analyzing differences between groups. The significance level was set to p < 0.05. At 1 DPI the snake venom induced extensive myonecrosis (Fig. 1A, E, K) and sarcolemmal disruptions evidenced by EBD fluorescence in both strains (Fig. 1I, J). Serum CK levels at 3 h after venom injection (Fig. 1L) confirmed that the extension

of acute tissue damage is similar in gastrocnemius muscle from C3H/HeJ mice with a non-functional TLR-4 receptor and C3H/HeN mice with functional receptor. Myonecrosis and intense inflammatory infiltration (3 DPI) corresponded nearly to 30% of the total tissue area in both C3H/HeJ and C3H/HeN (Fig. 1B, F, K). TLR4-deficient mice showed at 10 DPI a 3-fold (p < 0.05) increase in the area of injury compared to C3H/HeN mice ( Fig. 1C, (-)-p-Bromotetramisole Oxalate G, K). C3H/HeJ lesion was characterized by intense inflammatory infiltrate and connective tissue deposition ( Fig. 1C, G). No significant difference was observed in the CK activity between both strains ( Fig. 1K). At 21 DPI both strains showed ( Fig. 1D, H, K) numerous myofibers with central nucleation, an indication of efficient muscle regeneration. Regional lymph nodes from C3H/HeJ and C3H/HeN showed at 3 DPI similar increase of cellularity in the draining lymph nodes from venom inoculated muscles in comparison to the contralateral lymph node (Fig. 2A). However, at 10 and 21 DPI (Fig. 2B, C) TLR4-deficient mice showed a significant (p < 0.05, p < 0.001) increase of cellularity in the lymph node of the inoculated muscles compared to C3H/HeN wild-type mice. Intramuscular inoculation of the venom causes an increase of muscle mass due to massive edema formation (Barbosa et al., 2008).

New approaches are therefore needed to elucidate the structural features associated with bitterness CDK phosphorylation of amino acids and peptides, and to devise strategies to reduce the bitter sensation, including spray-drying encapsulation with maltodextrin and cyclodextrin as carriers [53], addition of bitter masking or inhibiting ingredients [54●], or enzymatic exopeptidase treatment [55]. Sensory-guided fractionation,

involving multi-step separations followed typically by mass spectrometry and ‘sensomics mapping’, is a recognized approach to identify the peptide sequences responsible for undesirable bitter taste of protein hydrolysates and their fractions [47], but requires evaluation by humans to verify the taste of individual peptides in the isolated fractions buy SCH772984 or chemically synthesized peptides. Not only is this a time-consuming and expensive process, there are technological challenges related to the small quantities of peptides typically available, as well as safety concerns for taste evaluation considering the non-food grade solvents and chemical reagents used in peptide

synthesis, fractionation and purification. Panelist fatigue, the limited number of samples that can be evaluated at a time, and difficulty with standardization particularly over long periods of time are also important considerations. QSAR may be useful to complement human sensory evaluations by providing clues to elucidate the bitterness of food-derived bioactive peptides 24•, 25, 56, 57 and 58, but, as previously mentioned, the validity and usefulness of the QSAR approach hinges on the information available for building the prediction model. Although human sensory evaluation will always be the ‘gold standard’ for assessing taste attributes and acceptability, in view of the aforementioned limitations, there has been increasing interest to develop instrumental taste sensing systems and electronic tongues for screening of large numbers of fractions and samples,

thus lightening the burden of human taste panel evaluation 59 and 60. Instrumental sensors have been applied to analyze taste of amino acids, pheromone peptides and protein hydrolysates 61, 62, 63 and 64••, and to assist in screening for compounds to mask their bitterness [65]. Cell based assays also show promise as an alternative to human panelists for screening of peptides for bitter taste. Using engineered cell lines expressing the TAS2R and the chimeric G protein α-subunit (Gα16gust44), positive interaction of peptides with the TAS2R receptor is detected fluorometrically by an influx of extracellular calcium indicator that is taken to represent activation by bitter peptides [51●].

Since then, the species has managed to colonise not only the Gulf of Finland but also waters farther west (the Gulf of Riga). Its abundance has been gradually increasing – ca ten-fold in six years. The maximum abundance was observed in 2004 (157 indiv. m− 3) ( Rodionova & Panov 2006), and in 2006 it was 120 indiv. m− 3 ( Põllupüü et al. 2008).

The Veliparib molecular weight results of the present study demonstrate the further expansion of E. anonyx. This Ponto-Caspian species was found in the Gulf of Gdańsk for the first time in July 2006. It was observed in the eastern and western Gulf of Gdańsk down to a maximum depth of 20 m until the second half of August in rather low densities, not exceeding 2 and 6 indiv. m− 3 in July and August respectively. A similarly low abundance, less than 1 indiv. m− 3, and the occurrence of the species in shallow waters was recorded at the beginning of the invasion of E. anonyx in the

Gulf of Finland ( Rodionova and Panov, 2006 and Põllupüü et al., 2008). We consider it probable that a period of seven years elapsed between the first record of E. anonyx in the Baltic Sea and the first one in the Gulf of Gdańsk. This is the same period of time as in the case NVP-BEZ235 chemical structure of the expansion of another cladoceran alien to the Baltic Sea, Cercopagis pengoi, which appeared for the first time in the Gulf of Riga in 1992 ( Ojaveer & Lumberg 1995) and in the Gulf of Gdańsk in 1999 ( Bielecka et al., 2000 and Duriš et al., 2000). In addition, at the beginning of its expansion into the Gulf of Gdańsk the E. anonyx population seemed to be Nintedanib (BIBF 1120) rather unevenly distributed – it was not recorded at three stations: So2, K2 and K4. A similar trend was observed in the case of C. pengoi – initially the species was recorded rather irregularly (Bielecka & Mudrak 2000). In the eastern Baltic Sea E. anonyx is most abundant in summer, in June and July ( Rodionova and Panov, 2006 and Põllupüü et al., 2008). Generally, E. anonyx was recorded in the eastern Gulf of Finland at a water temperature of 11–24.5° C and a salinity of 1–3

PSU, with the first specimens appearing at 17–18° C ( Rodionova & Panov 2006). However, other results were obtained by Põllupüü et al. (2008), who investigated a larger part of the Gulf of Finland (Tallinn Bay and Narva Bay) and the Gulf of Riga (Pärnu Bay). These authors found that E. anonyx was constantly present in the plankton when the water temperature reached 15 °C, with its maximum density being recorded at 16–20° C and 12–13 PSU ( Põllupüü et al. 2008). In the Gulf of Gdańsk E. anonyx was observed somewhat later in the year. It appeared at the beginning of July when the water temperature was 11.4–23.6 °C and was present for a shorter period of time – only a month and a half. Its abundance was correlated positively with water temperature. The ranges of water temperature and salinity during the occurrence of this cladoceran were 4.2–23.6° C and 4.6–7.5 PSU respectively. As indicated in an earlier study, E.

This volume will be referred to as the “RO-reviewed

TES CTV,” which is used to produce the planning target volume (PTV). For the purposes of comparing the dosimetric effect of the RO modifications, a second PTV was also generated directly from the Raw TES CTV, which will be referred to as the “Raw TES PTV.” The guidelines for the creation of the PTV at this institution recommend applying 0.3–0.5 cm lateral, 0–0.3 cm anterior, and 0.5 cm superior Bleomycin purchase margins to the CTV. No planning margins are added posteriorly or inferiorly to spare the rectum and penile bulb. Although small variations in the size of the margins were present among clinically generated PTVs, the margins applied to generate the Raw TES PTVs for this study complied with the guideline recommendations (0.3 cm lateral, 0.2 cm anterior, and 0.5 cm superior). An additional ATM inhibitor component of this study involved the use of contours that were generated completely manually (i.e., without the presence of any preliminary contours on the image sets) by multiple blinded observers (ROs, radiation therapists, and/or individuals trained by experts). We will describe these contours and their derivative structures as “manually”

generated to distinguish them from the “RO-reviewed TES” contours, which are informed by the TES algorithm. Brachytherapy treatment plans were developed for the PTVs by a single medical physicist. These plans adhered to the standard BCCA planning algorithm, which can be generally described as following a Vitamin B12 low-activity (0.424 U) modified peripheral loading strategy using custom-loaded, stranded seeds (RAPIDStrand; Oncura, Arlington

Heights, IL). Each plan is designed to provide 97% or higher coverage of the PTV and 99% or higher coverage of the CTV by the 100% (144 Gy) isodose, with a CTV V150 between 56% and 65% and PTV V150 between 50% and 60%. The V150 is geometrically biased to the posterolateral aspects of the target. The volume that does not reach prescription dose in planning is confined to a small region of the anterior base of the PTV whenever possible. To evaluate the TES method, two types of comparisons were carried out: volumetric and dosimetric. The volumetric comparisons aimed at evaluating the spatial agreement between Raw TES and RO-reviewed TES contours. The dosimetric comparisons were designed to investigate what the impact on coverage of the RO-reviewed PTV would have been if planning had been performed directly on the Raw TES PTV. To do this, treatment plans were originally created on Raw TES contours, while satisfying the BCCA planning goals, and subsequently superimposed on the corresponding RO-reviewed TES contours. Plans derived from Raw TES PTVs were also compared with the plans created on the manual contours of different observers on the same image set. Details of each of the evaluation methods are described in the next section. We will first define the evaluation measures used in this article.

The pairing of heavy and light chain V-genes from each family occurs in proportion to their abundance in the library (data not shown), indicating random pairing as expected with the library construction Quizartinib method that was employed.

Previous data suggests random pairing also occurs in the human repertoire (de Wildt et al., 1999). Each library was assessed by selection against seven targets: gastrin (a 14 amino acid peptide), β-galactosidase (a bacterial protein, β-gal), human proteins insulin receptor in complex with insulin (InsR + Ins), TIE1, TIE2, TIE2 in complex with angiopoeitin 1 (ANG1), and TIE2 in complex with angiopoeitin 2 (ANG2). Three rounds of panning were performed for each target using previously described panning methods (Hoet et al., 2005 and Bhaskar et al., 2012). For each target, five to ten 96-well buy CYC202 plates of clones were screened either by ELISA (gastrin, β-gal, TIE1, TIE2, and TIE2 complexes) or flow cytometry (InsR + Ins (Bhaskar et al., 2012), TIE2, and TIE2 complexes) for binding to the target. The clones that bound to their target were sequenced to identify unique clones. The unique clones were then analyzed for VH and VL family representation (Fig. 4), for CDR3 length of the VH and VL, and to assess the germline representation in FR1–FR3 of the selected clones (Table 2). Once unique clones were identified for each

target, further Sitaxentan characterization of those clones was performed. For both libraries, the unique clones that bound to β-gal and TIE1 were prepared

as soluble antibody fragments in periplasmic extracts (PPE) and the KD (equilibrium dissociation constant) was determined using Biacore. For both targets, multiple antibody fragments with high affinities (single-digit nM to triple-digit pM) were identified. Table 2 lists the best KD identified for each target per library (Fig. S3). When screening the panning campaigns of TIE2 in complex with either of its ligands (ANG1 or ANG2), antibody fragments in PPE were screened by flow cytometry for binding to TIE2 or TIE2–ligand complex, and screened by ELISA for binding to ANG1 or ANG2. Binders in three categories emerged: single-protein binders (TIE2, ANG1 or ANG2), TIE2/ANG1-complex binders, or TIE2/ANG2-complex binders (Table 3). A subset of 10 Fab clones and 8 scFv clones that bound TIE2 was reformatted as IgG and the KD for each clone was determined with Biacore (Table 2 and Fig. S3). For clones from XscFv2, 6 out of 8 clones have KDs > 8 nM. For clones from XFab1, 9 out of 10 have KDs > 11 nM with two of these clones having KDs in the pM range (Fab09 = 800 pM and Fab10 = 500 pM). The sequences of the 591 unique selected clones for both libraries were compared to each other and aligned to the closest germline sequence.

In studies in vitro, Uaesoontrachoon et al. (2008) reported that OPN released by myoblasts served as a link between the inflammatory response Selumetinib price and myogenesis during the early phase of muscle regeneration and repair. Our findings corroborate the close relationship in timing between the second phase of OPN upregulation and the significant increase in myogenin expression initiated at 18 h, with peaks at 3 days and 7 days post-venom. Our results showed that

B. lanceolatus venom promoted connective tissue disorganization in the acute stage of envenoming followed by patches of intense collagen deposition 3–7 days post-venom. Fibrotic processes may represent a barrier for tissue revascularization and limit the access of important molecules or cells involved in tissue regeneration. The finding that the small diameter of regenerated fibers at 21 days post-venom was significantly lower than in time-matched controls suggests that fibrosis may have impaired complete regeneration. It is worth

mentioning that OPN has been pointed out as a pro-fibrotic promoter in hepatic and renal diseases ( Lorena et al., 2006 and Irita et al., 2008). In cardiac muscle dysfunction ( Singh et al., 2010) and skeletal and cardiac muscles of mdx mice ( Vetrone et al., 2009) the upregulation of OPN has been correlated with enhanced collagen synthesis and accumulation, whereas deletion of the OPN gene reduced fibrosis and improved regeneration. Our findings this website also showed two other interesting data: the expression of myogenin in the cytoplasm of myoblasts and myotubes instead of its usual expression in the nucleus, and the population of CD68 + macrophages significantly elevated in the proliferative stage of myoblasts (3 days post-venom), and in the acute inflammatory phase (3–6 h post-venom). Nuclear myogenin is needed for regulation of the transcription of specific myogenic promoters whereas its retention in the cytoplasm may Enzalutamide molecular weight regulate the biological activity of proteins and prevent differentiation;

the transfer of myogenin into the nucleus occurs when proliferative signals cease and the protein level increases significantly (Ferri et al., 2009). On the other hand, macrophages can release products that inhibit the transition of myogenic cells from proliferative to differentiating stages (Merly et al., 1999). Whether this significant presence of phagocytic M1 macrophages on day 3 post-venom has a role in the atypical retention of myogenin in the cytoplasm and in delayed muscle repair is unknown. This is an interesting possibility since it was only from day 14 post-venom onwards that myogenin labeling was no longer observed in the cytoplasm and that CD68 macrophage numbers were as low as in control muscle.

This finding is in line with previous research done on trends in N AZD6244 mouse and P in Estonian rivers (Iital et al., 2005). Iital et al. (2005) found a downward trend in the amount of N in 91% of the studied sites while a downward

trend in the amount of P was observed in only 9% of the studied sites while also some upward trends were observed (9% of the studied sites). Table 2 and Fig. 3 show that there is a lot of variability between the catchments in both east and west. The regional variation in trends can prove interesting for management strategies that aim to reduce nutrient loads into the Baltic Sea. The focus should be more on catchments experiencing an increasing trend or no trend at all. Previous modelling studies projecting future changes of nutrient loads into the Baltic Sea focused on the entire basin-scale (Arheimer et al., 2014, Donnelly et al., 2014, Meier et al., 2012 and Meier et al., PTC124 2014). These modelling studies and their findings are often considered by policy makers to implement management strategies. Since our study demonstrates that large variation exists among the catchments, it can be suggested to look more at catchment-scale interactions when developing management strategies. This might reveal additional information which can lead to more focused and effective approaches to reach targeted reductions. The results

in Table 2 show the potential for nutrient reductions in the BSDB. Upscaling the 0.13 kg km−2 yr−2 reduction in TP observed in 13% of the total BSDB area (east + west) results in a potential reduction of 223 tonnes per year. Considering a similar upscaling for TN, there is a potential reduction of 10,980 tonnes per year. Target reductions GNA12 set by the Baltic Sea Action Plan (BSAP) correspond to a reduction of 135,000 tonnes TN and 15,250 tonnes TP by 2021 (HELCOM, 2007). If we assume these potential reduction rates for TN and TP, then the target reduction of TP would be reached in 68 years while the target reduction of TN would be reached in 12 years. Although it is unlikely that change rates calculated for the year 1970–2000 will be the same for 2000–2021, these

estimates suggest it is possible to reduce TP in the BSDB but that it will be difficult to reach the target reductions by 2021 without significant shifts in land management. Table 2 and Fig. 3 also show that the focus for management strategies should be more on P reduction rather than on N reduction as more catchments show an increasing trend rather than a negative trend for TP. This suggestion is further reinforced when the N:P ratio is taken into account. The N:P ratio steadily declined in eastern catchments from 30 to 16, which will ultimately affect the N:P ratio for the whole Baltic Sea. The results presented in this study suggest that a declining trend in N:P ratio is largely caused by an increase of TP from eastern catchments.

Positive tendencies of the recurrence of daily heavy precipitation

events were determined in the whole of Lithuania. However, it is quite difficult to distinguish the regions where the changes are the most intensive. According to the Mann-Kendall test significant changes were observed at separate meteorological stations representing different regions of Lithuania (Figure 6a). The recurrence trends of 3-day heavy precipitation are less clear and significant. Despite the prevailing positive tendencies (Figure 6b), at some locations the changes were negative. The number of cases when 3-day precipitation exceeded 20 mm varied from 0 in 1979 (Vilnius) to 20 in 1980 (Telšiai). An important indicator of an extreme precipitation event is the percentage of heavy precipitation Alectinib supplier in the total Z-VAD-FMK nmr annual amount. Mean percentages of daily heavy precipitation vary from 33 to 44% in Lithuania and can approach 60% in some years. The average 3-day heavy precipitation percentage varies from 27 to 41% and can exceed 60% in single years. In summer and autumn, the percentage of heavy precipitation is much higher than during the rest of the year. Analysis of the dynamics shows positive but mostly insignificant tendencies in a large part of Lithuania during the study period. This means that during recent decades the temporal unevenness of precipitation has increased.

This tendency is especially clear in the summer months, when extreme precipitation events increase against the background of neutral or negative trends in the total summer precipitation. An increase in daily and 3-day annual maximum values was determined during the study period. Moreover, positive tendencies of the mean annual precipitation maximum were calculated for all meteorological stations by splitting the 1961–2008 year period into two parts (Figure 7). The period from the middle

of the 1980s to the end of the 1990s was very abundant in heavy precipitation events. Long-term variability data from neighbouring countries are quite similar to our findings. In the western part of Russia an increase in the number of days with heavy precipitation was recorded in 1936–2000 (Bogdanova et al. 2010). There was also an increase in the number of days with heavy precipitation and in the DCLK1 intensity of heavy precipitation in 1925–2006 in Latvia (Avotniece et al. 2010). Most of the positive significant trends were observed in the cold season, particularly in winter, and no overall long-term trend in extreme precipitation was detected in summer (Lizuma et al. 2010). In Estonia there is a rising trend of extreme precipitation events (Tammets 2010) and of the total number of extremely wet days (Tammets 2007) in 1957–2006. In contrast, another study did not reveal any significant long-term trend of heavy precipitation in 1961–2005 in Estonia (Mätlika & Post 2008).

Activation of the SA pathway has been proven to be important in both basal and resistance gene (R)-mediated biotrophic pathogen defense in Arabidopsis thaliana, while the JA/ET pathway is activated in response to necrotrophic pathogens, feeding by tissue-damaging herbivores, and wounding [35]. Potato responses to infestation by aphids, a kind of sucking insect whose feeding behavior is similar to SBPH, involve both SA and JA/ET plant defense signaling pathways [36]. Another study showed that tomato

leaves rapidly accumulated high levels of SA after exposure to the cotton bollworm, a type of chewing pest [10]. Plants are usually exposed to insects and pathogens and hence have developed resistance to simultaneous pathogen infection and insect feeding. Angiogenesis inhibitor As insect damage can often increase the risk of pathogen attack this coordination

of plant responses seems to make biological sense. In the long-term evolutionary process, the SA- and JA-mediated signal transduction pathways have both been preserved [37]. Plants accurately regulate the SA and JA signaling pathways by adjusting SA and JA contents in order to resist stress more efficiently. In this study, the transcription of the key genes PAL for the SA synthesis pathway, as well as LOX and AOS2 for the JA pathway, were LGK-974 purchase significantly up-regulated compared with their basal levels, which indicated two signaling pathways were activated due to SBPH attack. The expression of PAL dramatically increased in Kasalath after SBPH sucking, which promoted synthesis of SA and then increased SA content. Therefore, the SA mediated signaling pathway was the major defense mechanism Branched chain aminotransferase in resistant Kasalath, which was consistent with the reports mentioned above [7], [10], [12], [15] and [31]. However, the induction LOX and AOS2 in JA responsive pathway in the susceptible Wuyujing 3 was somehow contradictory to the findings reached by Zanate et al. [15] As mentioned above, the JA/ET pathway usually induces genes whose protein products have antimicrobial and antifungal activity and accumulate

in response to necrotrophic pathogens [38]. In a previous study, we detected that wound healing was probably caused by some substance secreted by a resistance rice variety, which then protected the infected seedling. This substance was observable with a scanning electron microscope (SEM) on epidermis of resistant rice leaves infested by SBPH but not in the leaves of a susceptible variety [39]. Non-healing wounds caused by SBPH sucking in the susceptible genotype Wuyujing 3 might have led to a large invasion of bacteria and fungi in this genotype that did not occur in Kasalath which healed its wounds quickly. The massive accumulation of microorganisms in Wuyujing 3 was likely to significantly induce the expression of LOX and AOS2 involved in JA-mediated signal pathway.

, 2004, Kolko et al., 2007 and Bazan et al., 1986). Under abnormal conditions PLA2 and its products are involved in neuroinflammation processes, oxidative stress and neural cell injury. Furthermore, PLA2 and metabolites in excess are also related with Alzheimer’s and Parkinson’s diseases, ischemia and in multiple sclerosis (Farooqui and Horrocks, 2006). In brain, PLA2s mediate several

physiological responses: neurotransmitters release, neurite outgrowth, cellular membrane repair and cellular growth and differentiation (Farooqui and Horrocks, 2004). Based on these last results, the aim of this work was to analyze the effect of a PLA2 isolated from Lachesis muta snake venom (named LM-PLA2-I) on axotomized retinal ganglion cell survival kept in culture and also to investigate the mechanism of action of this PLA2 in cell survival. Medium 199 and fetal calf serum (FCS) were purchased from see more Gibco (Gaithersburg, USA), L. muta snake HSP inhibitor venom, 1.2-bis (2-aminophenexy) ethane-N,N,NX,NX-tetraaceticacid (BAPTA-AM), glutamine,

penicillin, lysophosphatidylcholine from egg yolk (L 4129), fatty acids mixture (1892-1AMP and 49441-U), streptomycin, p-bromophenacyl bromide (p-BPB), poly-l-ornithine and horseradish peroxidase (HRP) were obtained from Sigma (St. Louis, USA). Chelerythrine chloride came from BIOMOL (Plymouth Meeting, USA). Dimethyl sulphoxide (DMSO) was obtained from Mallinckrodt Baker (New Jersey, USA). The inhibitor of JNK V (1,3-Benzothiazol-2-yl-(2-((2-(3-Pyridinil)ethyl)amino)-4-pyrimidinyl)acetonitrile) and Rottlerin were purchased from Calbiochem (California, USA). Trypsin was obtained from Worthington Biochemical (New Jersey, USA). The phospholipase A2 enzyme (LM-LPA2-I) was purified by two chromatographies steps; a gel filtration on a Sephacryl S-200HR followed by a reverse-phase C2/C18 using FPLC apparatus as previously described (Fuly et al., 1997). Lipids (LPC as well as fatty acids) were dissolved in small volumes of chloroform, dried to a thin film under a gentle nitrogen

flow and Arachidonate 15-lipoxygenase vacuum pumped to remove the organic solvent. After, dried lipid was suspended in 150 mM NaCl, extensively vortexed and then ultrasonically dispersed in a bath sonicator for 5 min at room temperature. Then, lipids were kept at 4 °C until experiments. The enzymatic activity of LM-LPA2-I was measured by the indirect hemolysis method, by using washed rabbit erythrocytes and hen’s egg yolk as substrate, as previously described (Fuly et al., 1997 and Fuly et al., 2002). Modification on histidine residues of LM-LPA2-I was carried out by incubating PLA2 with p-bromophenacyl bromide (10 μM, final concentration) dissolved in DMSO. Incubation was carried out overnight at 4 °C, and then the excess of reagent was removed by dialysis, when necessary. Immediately after, samples were assayed for the indirect hemolytic test and the effect on retinal ganglion cell survival.