Isolation and functional characterization of proinflammatory acidic phospholipase A2 from Bothrops leucurus snake venom

AbstractIn the present study, an acidic PLA2, designated Bl-PLA2, was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA2 was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000Da and pI was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA2s from snake venoms. Its specific activity was 159.9U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA2 induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-α, IL-1β and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA2 induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism

Subcellular localization and kinetic characterization of a gill (Na +, K+)-ATPase from the giant freshwater prawn macrobrachium rosenbergii

The stimulation by Mg2+, Na+, K+, NH 4 +, and ATP of (Na+, K+)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na +, K+)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M r. Under saturating Mg2+, Na+, and K+ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V M = 115.0 ± 2.3 U mg-1, K 0.5 = 0.10 ± 0.01 mmol L-1. Stimulation by Na+ (V M = 110.0 ± 3.3 U mg-1, K 0.5 = 1.30 ± 0.03 mmol L -1), Mg2+ (V M = 115.0 ± 4.6 U mg -1, K 0.5 = 0.96 ± 0.03 mmol L-1), NH4 + (V M = 141.0 ± 5.6 U mg -1, K 0.5 = 1.90 ± 0.04 mmol L-1), and K+ (V M = 120.0 ± 2.4 U mg-1, K M = 2.74 ± 0.08 mmol L-1) followed single saturation curves and, except for K+, exhibited site-site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K I = 12.4 ± 1.3 mol L-1. Complementary inhibition studies suggest the presence of F0F1-, Na+-, or K +-ATPases, but not V(H+)- or Ca2+-ATPases, in the gill microsomal preparation. K+ and NH4 + synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4 +, and K+ of the gill enzyme. © 2013 Springer Science+Business Media New York

Modulation by K+ Plus NH4+ of microsomal (Na+, K+)-ATPase activity in selected ontogenetic stages of the diadromous river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

We investigate the synergistic stimulation by K(+) plus NH4 (+) of (Na(+), K(+))-ATPase activity in microsomal preparations of whole zoea I and decapodid III, and in juvenile and adult river shrimp gills. Modulation of (Na(+), K(+))-ATPase activity is ontogenetic stage-specific, and particularly distinct between juveniles and adults. Although both gill enzymes exhibit two different sites for K(+) and NH4 (+) binding, in the juvenile enzyme, these two sites are equivalent: binding by both ions results in slightly stimulated activity compared to that of a single ionic species. In the adult enzyme, the sites are not equivalent: when one ion occupies its specific binding site, (Na(+), K(+))-ATPase activity is stimulated synergistically by ≈ 50% on binding of the complementary ion. Immunolocalization reveals the enzyme to be distributed predominantly throughout the intralamellar septum in the gill lamellae of juveniles and adults. Western blot analyses demonstrate a single immunoreactive band, suggesting a single (Na(+), K(+))-ATPase α-subunit isoform that is distributed into different density membrane fractions, independently of ontogenetic stage. We propose a model for the modulation by K(+) and NH4 (+) of gill (Na(+), K(+))-ATPase activity. These findings suggest that the gill enzyme may be regulated by NH4 (+) during ontogenetic development in M. amazonicum

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This paper focuses on the path of appearance and awareness

Effect of various inhibitors on ATPase activity in gill microsomes from juvenile and adult <i>M. amazonicum.</i>

<p>Assays were performed continuously at 25°C in 50 mmol L⁻¹ HEPES buffer, pH 7.5, containing 2 mmol L⁻¹ ATP, 5 mmol L⁻¹ MgCl₂, 20 mmol L⁻¹ KCl and 50 mmol L⁻¹ NaCl for juveniles or 20 mmol L⁻¹ NaCl for adults, in a final volume of 1.0 mL. Data are the mean ± SD from three (N = 3) different microsomal preparations. Oligomycin was prepared in ethanol. Bafilomycin and thapsigargin were prepared in dimethylsulfoxide.</p><p>*Significantly different from respective value for juvenile (P≤0.05).</p

Kinetic parameters for the stimulation by K⁺ and NH₄⁺ of (Na⁺, K⁺)-ATPase activity in whole zoea I and decapodid III, and juvenile and adult <i>M. amazonicum</i> gills.

<p>Initial rates were measured in 50 mmol L⁻¹ HEPES buffer, pH 7.5, containing 2 mmol L⁻¹ ATP, 5 mmol L⁻¹ MgCl₂, 50 mmol L⁻¹ NaCl, and the given concentrations of KCl and NH₄Cl, in a final volume of 1.0 mL. Data are the mean ± SD from at least three different larval or gill preparations.</p

Immunolocalization of the gill (Na⁺, K⁺)-ATPase α-subunit in juvenile and adult <i>M. amazonicum.</i>

<p>Frozen cross sections taken transversely to the gill lamellae long axes were incubated with mouse monoclonal IgG α-5 antibody raised against chicken (Na⁺, K⁺)-ATPase α-subunit then incubated in donkey anti-mouse IgG secondary antibody conjugated with Alexa-fluor 488. Phase contrast/DAPI/α-5 images demonstrating typical lamellar structure. Immunofluorescence labeling (Alexa-fluor 488, 495/519 nm) showing distribution of the (Na⁺, K⁺)-ATPase α-subunit (green) located predominantly in the intralamellar septal cells identified by their DAPI-stained nuclei (blue). <b>A-</b> Juvenile gill lamellae. <b>B</b>- Adult gill lamellae. Scale bars  = 50 µm.</p

Sucrose density gradient centrifugation of a microsomal fraction from gill tissue of juvenile and adult <i>M. amazonicum</i>.

<p>Aliquots containing 4.5% (w/w) continuous sucrose density gradients. Fractions (0.5 mL) collected from the bottom of each gradient were analyzed for total ATPase activity (○), (Na⁺, K⁺)-ATPase activity (•), ouabain-insensitive ATPase activity (▵), protein concentration (▴) and sucrose concentration (□). <b>Inset</b>: Adult gill tissue.</p

SDS-PAGE and Western blot analyses of microsomal fractions from whole decapodid III, and juvenile and adult <i>M. amazonicum</i> gills.

<p>Electrophoresis was performed in a 5–20% polyacrylamide gel using 4 μg microsomal protein for silver staining and 160 μg for Western blotting. The analysis was repeated three times (<i>N</i> = 3) using aliquots from different homogenates prepared from each ontogenetic stage. <b>Silver nitrate-stained gels</b>: <b>A-</b> Decapodid III. <b>B-</b> Juvenile. <b>C</b>- Adult. <b>Western blots</b>: <b>D-</b> Decapodid III. <b>E</b>- Juvenile. <b>F</b>- Adult.</p

Acute Toxicity of Schizolobium parahyba Aqueous Extract in Mice

The herbal extract of Schizolobium parahyba leaves is used commonly in the Brazil central region to treat snakebites. This study evaluates the acute toxicological effects of Schizolobium parahyba aqueous extract in mice 24 h after intraperitoneal administration. Acute toxicity was evaluated using biochemical, hematological and histopathological assays. Alterations in the levels of transaminases, bilirubin, albumin and prothrombrin time were observed, and these are likely to occur due to hepatic injury, which was confirmed by light microscopy. Liver histopathological analysis revealed the presence of lymph plasmocitary inflammatory infiltrate, but no other histopathological alterations were observed in any of the other organs analysed. The data confirm the low toxicity of the extract of Schizolobium parahyba and provide a model for the selection of a dose that does not cause injuries in the organism. Copyright (C) 2009 John Wiley & Sons, Ltd.FAPEMIGCNPqCAPE