Surface characterization and interfacial activity of chitinase chi18-5 against chitosan in langmuir monolayers.

One of the challenges for producing active chitinase formulations relies on the gap between the laboratory tests and the biological scenarios where the enzyme will perform its function. In this work, we have employed different Langmuir monolayer arrays to evaluate the interfacial behavior of a recently purified recombinant chitinase, Chi18-5. We have demonstrated that two conformations exist for the chitinase at pH values close to its pI, showing very distinct structural properties at the air/aqueous interface. Enzyme activity was assessed by implementing different kinetic approaches and using a chitosan-1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) mixed film as organized substrate model membrane. Combining these strategies, we demonstrated that better catalytic efficiencies can be obtained for Chi18-5 at pH 5. Moreover, the chitinase activity at the air/aqueous interface can be tuned by introducing in situ pH modifications over the surrounding milieu. We also studied the changes in the topography at the mesoscale level using Brewster Angle Microscopy (BAM). We found that Chi18-5 segregated onto the chitosan domains of the membrane, showing differences in homogeneity depending on the pH imposed. Alternatively, pure Chi18-5 was tested for immobilization onto a hydrophilic activated solid support using the Langmuir-Blodgett technique. Atomic Force Microscopy (AFM) analyses showed successfully stabilization and preservation of molecular features attributed to the pH at which the enzyme deposition was performed.

Reversible exposure of hydrophobic residues on albumin as a novel strategy for formulation of nanodelivery vehicles for taxanes.

BACKGROUND: We report herein a novel strategy for the preparation of protein-based nanodelivery vehicles for hydrophobic active pharmaceutical ingredients. METHODS: The procedure consisted of three steps, ie, exposure of hydrophobic residues of a protein to a pH-induced partial unfolding: interaction between hydrophobic residues on the protein and the hydrophobic active pharmaceutical ingredient, and a final step where the structure of the protein was reversed to a native-like state by returning to neutral pH. As proof of concept, the interaction of paclitaxel with partially unfolded states of human serum albumin was evaluated as a potential method for the preparation of water-soluble complexes of the taxane with albumin. RESULTS: We found that paclitaxel readily binds to pH-induced partially unfolded albumin, leading to the formation of optically clear water-soluble complexes. The complexes thus formed were more stable in solution when the albumin native state was at least partially restored by neutralization of the solution to a pH around 7. It was also observed that the hydrodynamic radius of human serum albumin was only slightly increased after the cycle of pH changes, remaining in a monomeric state with a size according to paclitaxel binding. Furthermore, paclitaxel binding did not affect the overall exposure of charged groups of human serum albumin, as evaluated by its interaction with an ionic exchange resin. CONCLUSION: The in vitro biological activity of the complexes formed was qualitatively equivalent to that of a Cremophor(®)-based formulation.

Polyisoprene matrix for progesterone release: in vitro and in vivo studies.

Latex, a polyisoprene (PI) hydrophobic elastomer, was evaluated in vitro and in vivo as a matrix for intravaginal steroid hormone delivery. Matrices containing hormone were prepared by swelling latex in chloroform that contained soluble progesterone (P4). In vitro studies demonstrate that P4 release from PI follows a zero order model during at least 100 h and depends on initial load up to 10 mg cm(-2). The release of P4 from a PI matrix was found to be two times faster than from a polydimethylsiloxane (PDMS) matrix. FT-IR and X-ray powder diffraction analysis of P4 polymorphs show that when nucleated in PDMS, the hormone crystallizes only in alpha-form while in latex, crystallizes as a mixture of alpha- and beta-form. In vivo studies show that devices with a PI matrix containing 0.5 g of P4 are effective to reach plasma levels above 1 ng ml(-1) that are needed to synchronize estrous in cattle. Altogether, the results show that PI, a vulcanized polymer with a carbon-carbon backbone, can be used as a new matrix for the intravaginal administration of progesterone with improved release profile than silicone and that the matrix can influence the crystalline state of the hormone.

Ability of the polysaccharide chitosan to inhibit proliferation of CD4+ lymphocytes from mucosal inductive sites, in vitro and in vivo.

OBJECTIVE: After oral administration of chitosan (a copolymer of glucosamine and N-acetylglucosamine), mesenteric lymph node (MLN) lymphocytes exhibited traits of anergy, a process coupled with inability of mature T cells to proliferate. We wondered whether biological activity of chitosan could be affecting division of lymphocytes at the mucosal inductive sites. MATERIALS AND METHODS: We studied the effect of chitosan on proliferation of carboxyfluorescein diacetate-labelled MLN lymphocytes stimulated with concanavalin A in vitro. We assessed expression of CD25 and CD71 activation markers and pro-apoptotic molecule CD95L. Moreover, we studied the effect of chitosan ex vivo, in carboxyfluorescein diacetate-labelled MLN cells isolated after feeding single or repetitive doses of the polysaccharide, and we evaluated cell cycle parameters. RESULTS: Chitosan suppressed cell proliferation and down-modulated expression of CD25 in these MLN CD4+ cells isolated from normal rats. After in vivo contact, chitosan inhibited proliferation of MLN cells and reduced secretion of interferon-gamma. Furthermore, sustained feeding produced reduction in percentage of CD4+ cells in S phase of the cell cycle. CONCLUSION: Here we demonstrate the ability of chitosan to suppress proliferation of CD4+ lymphocytes from mucosal inductive sites in vivo and in vitro This effect could be relevant in modulatory activity of chitosan in the intestinal microenvironment.

The biocompatible polysaccharide chitosan enhances the oral tolerance to type II collagen.

Chitosan is a mucoadhesive polysaccharide that promotes the transmucosal absorption of peptides and proteins. At mucosal sites chitosan exhibits immunomodulatory activities and stimulates the release of regulatory cytokines. Herein we evaluated the effect of the co-administration of chitosan in the tolerance to type II collagen (CII) using an experimental model of arthritis. Rats were fed diluent (acetic acid), 1 mg CII, 1 mg chitosan or 1 mg CII + 1 mg chitosan during 5 days before immunization with CII in Freund's complete adjuvant. Systemic effects were evaluated in draining lymph nodes after antigenic challenge or during the clinical evolution of arthritis. Specific antibodies, proliferation against CII and the production of interferon (IFN)-gamma and interleukin-10 were assessed. Clinical signs were observed 13-15 days after primary immunization. The CII : chitosan group presented the lowest incidence and developed moderate arthritis, with reduced levels of immunoglobulin (Ig)G2a anti-CII, a limited proliferation in draining lymph nodes and a lower release of IFN-gamma after restimulation with CII. Our results demonstrate that chitosan enhances the tolerance to an articular antigen with a decrease in the inflammatory responses and, as a consequence, an improvement in clinical signs.

Room temperature vulcanizing silicone sheaths on a reusable support for progesterone delivery in estrous synchronization treatments in cattle.

High temperature vulcanizing silicone elastomers have been widely used in controlled delivery systems of steroid hormones with the aim of controlling estrous cycle in livestock. This paper reports experiments conducted to evaluate the possibility of using room temperature vulcanizing (RTV) silicone elastomers for the intravaginal administration of progesterone to cattle. In vitro studies showed that RTV silicones and high-temperature vulcanizing silicone release progesterone at a similar rate. Y-shaped inserts made of different polymers were designed as supports of RTV silicone sheaths to test the in vivo release of progesterone. Field evaluation showed that RTV silicone sheaths containing 0.75 g of progesterone were at least as effective at estrous synchronization as commercially available intravaginal inserts.

Improvement of HDL- and LDL-cholesterol levels in diabetic subjects by feeding bread containing chitosan.

In this work we evaluated the efficacy and safety of a bread formulation containing chitosan in dyslipidemic type 2 diabetic subjects. For this purpose a total of 18 patients were allowed to incorporate to their habitual diets 120 g/day of bread containing 2% (wt/wt) chitosan (chitosan group, n= 9) or standard bread (control group, n= 9). Before the study and after 12 weeks on the modified diet, the following parameters were evaluated: body weight, plasma cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, triglyceride, and hemoglobin A(1c) (HbA(1c)). Compared with the control group, the patients receiving chitosan-containing bread decreased their mean levels of LDL-cholesterol and significantly increased their mean levels of HDL-cholesterol at the end of the study. There were no significant differences in the body weight, serum triglyceride, and HbA(1c). These results suggest that chitosan incorporated into bread formulations could improve the lipoprotein balance similar to typical biliary salts trappers, increasing the HDL- and lowering the LDL-cholesterol, without changing the triglyceride levels. These results warrant further studies over a longer period of time to evaluate if a persistent improvement in levels of lipoproteins can be attained with this strategy.

Treatment of rheumatoid arthritis by oral administration of bovine tracheal type II collagen.

We evaluated the efficacy and safety of orally administered bovine tracheal type II collagen (CGII) in the treatment of rheumatoid arthritis (RA). Twenty RA patients received 0.5 mg/day of CGII for 12 weeks. Eighteen of them had improvements in the clinical parameters studied (swollen and tender joint counts, 15-m walking time, duration of morning stiffness, and physician's global assessment of disease activity). Anti-CGII antibodies were positive in 57% and rheumatoid factor (RF) in 71% of the patients with a short history of RA ( < or =2 years), whereas only 23% of those with long histories (>2 years) presented autoantibodies to CGII and 38% had positive RF. After the treatment, four patients showed reduced RF levels and all those with detectable serum tumor necrosis factor alpha (TNF-alpha) experienced its return to normal or levels below those at study entry. Although a placebo effect cannot be discounted, the oral administration of bovine tracheal CGII induced clinical benefits in 90% of the patients, without the side effects usually associated with treatment. This is the first study showing that feeding CGII can induce reductions in RF and TNF-alpha. The data justify further controlled studies to assess the long-term efficacy of this treatment approach.

Characterization of casein micelle precipitation by chitosans.

We have found that the addition of chitosan, a cationic polymer, on whole or skim milk produces destabilization and coagulation of casein micelles that takes place without changes in the milk pH or the stability of most whey proteins. The amount of lipids recovered in the chitosan-casein aggregates was similar or higher than that obtained with rennet or acid precipitation. Approximately 70% of milk Ca2+ (approximately 750 mg/L) was found in the chitosan-induced aggregates, which is 10 and 50% higher than the amounts observed with acid or rennet coagulations, respectively. Purified alpha, beta-, and kappa-caseins were extensively precipitated by different molecular weight chitosans at pH 6.8. The phosphate groups of caseins seem not to be relevant in this interaction because dephosphorylated alpha- and beta-caseins were equally precipitated with chitosans. Analysis by optical microscopy of the chitosan-casein complex reveals that the size of the aggregates increase as the molecular weight of chitosans increase. Hydrophobic and electrostatic interactions particpate in the association and coagulation of casein micelles with chitosans of different molecular weights. The phenomenon is observed over a broad range of temperature (4 to 70 degrees C) with a reduction in the concentration of chitosan needed to precipitate the caseins that parallels a reduction in the viscosity of the chitosan solutions. Taken together, the results indicate that the electrostatic interactions may contribute energetically to the association between the two biopolymers, but the hydrophobicity of the complex would be the key determinant in the overall energetics of the reaction.

Hydrolysis of a chitosan-induced milk aggregate by pepsin, trypsin and pancreatic lipase.

The addition of chitosan to whole milk results in dose dependent destabilization and coagulation of the casein micelles and milk fat. The present study evaluates how the presence of chitosan could affect the hydrolysis of this chitosan-induced aggregate by different gastrointestinal proteases (pepsin and trypsin) and by pancreatic lipase. The chitosan-milk aggregate was hydrolyzed by pepsin and trypsin, as evaluated by the UV absorbance of TCA-soluble peptides and by urea-PAGE. The kinetics and extent of hydrolysis were independent of the casein being soluble or aggregated. The release of soluble peptides from the aggregate was independent of the presence of chitosan. A progressive inhibition of pancreatic lipase was observed in proportion to the increase in molecular weight of the chitosan employed to induce the formation of the aggregate. Interestingly, the presence of chitosan not only affected the initial velocity of the reaction, but also reduced its extent. The results indicate that a milk aggregate induced by chitosan was very well digested by gastric and intestinal proteases independently of the molecular weight of the chitosan used, and that the aggregate could retain the lipid-lowering effect of chitosan.

Inhibition of human platelet aggregation by gangliosides.

The content and composition of gangliosides is modified upon platelet stimulation, suggesting that these lipids may play functional roles in platelet physiology. Therefore, the effect of exogenously added gangliosides on human platelet aggregation was evaluated. The pretreatment of platelets with a mixture of total gangliosides from bovine brain and a series of purified mono-, di- and tri-sialogangliosides partially inhibit the collagen-induced aggregation process and ATP release and completely block the generation of the second aggregation wave when ADP is used as agonist. The inhibition was exerted at around 100 microM by G(TOT) as well as purified G(M1), G(M3), G(D1a), and G(T1b) gangliosides, whereas asialoG(M1) and sulphatide did not show a significant influence on platelet aggregation. Thrombin, Ca(2+) ionophores (A23187 and Ionomycin), arachidonic acid, and U46619 were unable to bypass the inhibitory effect exerted by gangliosides, suggesting that gangliosides inhibit platelet aggregation by inhibiting the synthesis or action of prostaglandins. Gangliosides inhibited U46619-induced aggregation, thus suggesting that they block the action of thromboxane A(2). Epinephrine induces a partial aggregation on gangliosides-treated platelets, similar to fluoroaluminate and phorbol myristate acetate, indicating that these platelets are still functional. To summarize, these results indicate that the major pathway(s), but not all, driving to the aggregation process following the interaction of ligand-receptor may be blocked by pretreatment of human platelets with gangliosides.

Chitosan-induced phospholipase A2 activation and arachidonic acid mobilization in P388D1 macrophages.

We have found that chitosan, a polysaccharide present in fungal cell walls, is able to activate macrophages for enhanced mobilization of arachidonic acid in a dose- and time-dependent manner. Studies aimed at identifying the intracellular effector(s) implicated in chitosan-induced arachidonate release revealed the involvement of the cytosolic Group IV phospholipase A2 (PLA2), as judged by the inhibitory effect of methyl arachidonoyl fluorophosphonate but not of bromoenol lactone. Interestingly, priming of the macrophages with lipopolysaccharide renders the cells more sensitive to a subsequent stimulation with chitosan, and this enhancement is totally blocked by the secretory PLA2 inhibitor 3-(3-acetamide)-1-benzyl-2-ethylindolyl-5-oxy-propanesulfonic acid (LY311727). Collectively, the results of this work establish chitosan as a novel macrophage-activating factor that elicits AA mobilization in P388D1 macrophages by a mechanism involving the participation of two distinct phospholipases A2.

Calcium dependency of arachidonic acid incorporation into cellular phospholipids of different cell types.

Ca2+ -independent phospholipase A2 (iPLA2) is involved in the incorporation of arachidonic acid (AA) into resting macrophages by the generation of the lysophospholipid acceptor. The role of iPLA2 in AA remodeling in different cells was evaluated by studying the Ca2+ dependency of AA uptake from the medium, the incorporation into cellular phospholipids, and the effect of the iPLA2 inhibitor bromoenol lactone on these events. Uptake and esterification of AA into phospholipids were not affected by Ca2+ depletion in human polymorphonuclear neutrophils and rat fibroblasts. The uptake was Ca2+ independent in chick embryo glial cells, but the incorporation into phospholipids was partially dependent on extracellular Ca2+. Both events were fully dependent on extra and intracellular Ca2+ in human platelets. In human polymorphonuclear neutrophils, the kinetics of incorporation in several isospecies of phospholipids was not affected by the absence of Ca2+ at short times (<30 min). The involvement of iPLA2 in the incorporation of AA from the medium was confirmed by the selective inhibition of this enzyme with bromoenol lactone, which reduced < or =50% of the incorporation of AA into phospholipids of human neutrophils. These data provide evidence that suggests iPLA2 plays a major role in regulating AA turnover in different cell types.

A new phospholipase A2 isoform isolated from Bothrops neuwiedii (Yarará chica) venom with novel kinetic and chromatographic properties.

A new phospholipase A2 isoform, called P-3, isolated from Bothrops neuwiedii (Yarará chica) venom, showed different chromatographic, enzymatic and cytotoxic properties compared to the previously purified isoforms P-1 and P-2 but it had a similar edema-inducing activity. In contrast to previously reported B. neuwiedii phospholipase A2 isoforms, P-3 did not interact with the oligosaccharide matrix of gel filtration columns (Superose, Superdex). Its molecular weight was 15,000 and its N-terminal 14 amino acid sequence was Asn-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Lys-Ile-Ala-Gly. Amino acid analyse revealed the presence of an unique histidine, presumably located at the active site, because a full inhibition of enzymatic activity was observed after treatment with p-bromophenacyl bromide. The new isoform also differentiated in its surface pressure activity profile when assayed in lipid monolayers. P-3 had an optimum activity towards dilauroylphosphatidylcholine monolayers of 27 mN/m and a cut-off pressure of 30 mN/m, whereas P-1 and P-2 had an optimum of 13 mN/m with a cut-off of 22 mN/m. P-3 retained its edema-inducing activity in the absence of hydrolytic activity, suggesting that the inflammatory activity was not dependent on the enzymatic activity. Neither the enzymatic nor the edema-inducing activity was affected by heparin. The new isoform was not lethal when a single dose of 5 micrograms/g body weight was injected intraperitoneally into mice. All of the isoforms displayed cytotoxic activity in vitro on B16F10 melanoma cells evaluated by direct MTT assay, with an EC50 of 31 micrograms/ml for P-3 and of 15 micrograms/ml for P-1 and P-2. The cytotoxic activity of P-3 was inhibited by p-bromophenacyl bromide treatment of the enzyme (up to 170 micrograms/ml), whereas the same treatment on P-1 and P-2 changed their EC50 to 60 micrograms/ml. The difference observed with inhibited enzymes suggests a different mechanism for the cytotoxic action of P-3 with respect to P-1 and P-2.

Modulation of the inflammatory response by corticotropin-releasing factor.

Peptides of the corticotropin-releasing factor (CRF) family have been shown to have either pro- or anti-inflammatory activities. CRF (10-30 micrograms/kg) administered subcutaneously or intravenously could inhibit edema and dye leakage in the rat paw produced by several injuries. These findings are opposed to some results suggesting a predominantly pro-inflammatory effect of CRF mainly in arthritic processes. The purpose of this work was to identify in vivo and in vitro the conditions for the pro- or anti-inflammatory actions of CRF in order to clarify its physiological and pharmacological function. Using the rat paw edema test we observed that only the highest doses of CRF employed (5 micrograms) induced a moderate and sustained swelling. Pre-treatment with low doses of CRF (0.5-5 ng) was able to inhibit the edema induced by Naja naja phospholipase A2, carrageenin or histamine. Higher doses (50 ng-5 micrograms) had no anti-inflammatory activity. When co-inhibited with Naja naja phospholipase A2 or histamine the peptide did not modify the swelling at doses up to 500 ng, showing at 5 micrograms an additive edema with Naja naja phospholipase A2. In vitro, CRF did not modify the release of histamine but slightly increased the release of arachidonic acid to the medium. Our findings show a clear dose dependence on the local effects of CRF in inflammatory responses. These results suggest that the mechanisms of the two dose-related phenomena may be distinct.

Inhibition by gangliosides of Bacillus cereus phospholipase C activity against monolayers, micelles and bilayer vesicles.

The effect of complex glycosphingolipids (gangliosides) on the activity of phospholipase C from Bacillus cereus was studied using lipid monolayers, mixed micelles and small unilamellar vesicles containing phosphatidylcholine as substrate. In all artificial membrane systems assayed, gangliosides exhibit qualitatively similar inhibitory properties. Gangliosides decrease the enzyme activity irrespective of the aggregation structure in which the substrate is offered to B. cereus phospholipase C, and they do not affect the adsorption process of the enzyme. The modulatory effect of gangliosides occurs at the level of the interface, affecting both the maximum rate of catalysis of the enzyme already adsorbed and the availability of the substrate in a suitable organization for enzyme catalysis to take place.

Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages.

Cellular levels of free arachidonic acid (AA) are controlled by a deacylation/reacylation cycle whereby the fatty acid is liberated by phospholipases and reincorporated by acyltransferases. We have found that the esterification of AA into membrane phospholipids is a Ca(2+)-independent process and that it is blocked up to 60-70% by a bromoenollactone (BEL) that is a selective inhibitor of a newly discovered Ca(2+)-independent phospholipase A2 (PLA2) in macrophages. The observed inhibition correlates with a decreased steady-state level of lysophospholipids as well as with the inhibition of the Ca(2+)-independent PLA2 activity in these cells. This inhibition is specific for the Ca(2+)-independent PLA2 in that neither group IV PLA2, group II PLA2, arachidonoyl-CoA synthetase, lysophospholipid:arachidonoyl-CoA acyltransferase, nor CoA-independent transacylase is affected by treatment with BEL. Moreover, two BEL analogs that are not inhibitors of the Ca(2+)-independent PLA2--namely a bromomethyl ketone and methyl-BEL--do not inhibit AA incorporation into phospholipids. Esterification of palmitic acid is only slightly affected by BEL, indicating that de novo synthetic pathways are not inhibited by BEL. Collectively, the data suggest that the Ca(2+)-independent PLA2 in P388D1 macrophages plays a major role in regulating the incorporation of AA into membrane phospholipids by providing the lysophospholipid acceptor employed in the acylation reaction.

Identification of two specific lysines responsible for the inhibition of phospholipase A2 by manoalide.

Manoalide, a natural product of sponge, irreversibly inhibits phospholipase A2 (PLA2) by reacting with lysine residues. Cobra venom PLA2 mutants were constructed in which four of the six lysine residues were independently replaced by arginine or methionine, which cannot react with manoalide. The mutants were overexpressed in Escherichia coli, renatured, and purified. The enzyme mutants lacking Lys-6 (K6R and K6M) or Lys-79 (K79R) were inhibited only 40% by manoalide while the native cobra venom PLA2 was inhibited 80% under the same conditions. This means that the manoalide modification of either Lys-6 or Lys-79 accounted for only half of the manoalide inhibition. The double mutant (K6R79R) was not inhibited by manoalide at all. Lys-56 (K56R) and Lys-65 (K65R) mutants were inhibited to the same extent as the native enzyme which indicates that these residues are not responsible for any of the inhibitory effects produced by manoalide. These results demonstrate that the reaction of manoalide with both Lys-6 and Lys-79 can account for all of its inhibition of cobra venom PLA2. The inhibition of PLA2 and its mutants with manoalide did not affect the activity of the enzyme toward monomeric substrate, which suggests that manoalide does not modify the catalytic site residues, that it does not block access to this site, and that its inhibition requires an interface. Furthermore, as with native PLA2, the activation of phosphatidylethanolamine hydrolysis by phosphorylcholine-containing compounds was exhibited by all of the mutants suggesting that none of the lysines examined are essential for this activation.

Kinetic and pharmacologic characterization of phospholipases A2 from Bothrops neuwiedii venom.

Two phospholipases A2 (PLA2) (EC 3.1.1.4) were purified from Bothrops neuwiedii venom (isoenzymes P-1 and P-2). The molecular weights of P-1 and P-2 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 15,000 and 16,200 and the isoelectric points were 4.8 and 4.6, respectively. The N-terminal 14-amino-acid sequences determined were Asn-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Met-Ile-Ala-Gly and Ser-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Met-Ile-Ala-Gly for P-1 and P-2, respectively. Since both show sequence almost identical to that of a PLA2 from Crotalus atrox it was tentatively classified as being of group II. The enzymatic activity of P-1 and P-2 toward lipid monolayers was studied. The hydrolysis of dilauroylphosphatidylcholine (dlPC) shows a broad optimum between 7 and 18 mN m-1 and a cut-off pressure of 22 mN m-1. The activity toward dlPC displays a maximum at pH 8 and is dependent on the presence of Ca2+ with an apparent Kd of 0.1 mM, for both enzymes. P-1 and P-2 are heat-stable enzymes, unable to hydrolyze dilauroylphosphatidic acid monolayers. The enzymes are not lethal to mice at doses up to 5 micrograms/g body weight by intraperitoneal injection and they do not show myotoxic (up to 40 micrograms) or hemolytic activity (up to 8.5 micrograms/ml). Both lack anti-coagulant activity, determined by absence of changes in the recalcification time of platelet poor plasma (up to 100 micrograms/ml), and are not able to induce platelet aggregation (up to 50 micrograms/ml). However, both isoenzymes exhibit an important edema-inducing activity that is not altered at short times by the irreversible chemical inactivation of the hydrolytic activity with phenacyl bromide. P-1 and P-2 are able to release arachidonic acid from membrane phospholipids of neutrophils, property that is lost by the inactivation of the enzyme. This suggests that the edema-inducing activity of the active but not the inactive forms may be partly due to arachidonic acid-derived mediators. The edema-inducing activity of the active or inactive forms of the enzymes is inhibited by antagonists of histamine, suggesting that histamine plays an important role in both the active and the inactive B. neuwiedii PLA2s-induced edema. The results suggest that the inflammatory and the catalytic activity of these enzymes constitute separate properties.

Gangliosides raise the intracellular Ca2+ level in different cell types.

Total gangliosides from bovine brain at micromolar concentration induce intracellular Ca2+ increments in a temperature, time and dose dependent manner when assayed with suspensions of rat macrophages, rat and chicken neurons, human erythrocytes and liposomes, loaded with the fluorescent Ca2+ indicator FURA 2. The effect was independent on the endogenous ganglioside composition of the cells and in the case of neurons it was also independent on the differentiation state. Gangliosides do not induce the release of Ca2+ from inner stores. These findings indicate that the reported inhibition of arachidonic acid release (Bressler, J., et al., (1994) Life Sci., 54, 49-60) and anti-inflammatory properties of gangliosides (Correa, S.G. et al., (1991) Eur. J. Pharmacol. 199, 93-98) are not due to impairments of Ca2+ flux. The results also suggest the possibility that the well-known neurotrophic effect produced by gangliosides on undifferentiated neurons in culture may be due to subtoxic cytosolic Ca2+ increments.