Isolation and biochemical characterization of a γ-type phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum.

In the present work, we describe the isolation and partial structural and biochemical characterization of the first phospholipase A2 inhibitor (γPLI) from Crotalus durissus collilineatus (Cdc) snake serum. Initially, the Cdc serum was subjected to a Q-Sepharose ion exchange column, producing six peaks at 280 nm absorbance (Q1-Q6). Subsequently, Q4 fraction was submitted to affinity chromatography with immobilized PLA2 BnSP-7, a step that resulted in two fractions (NHS-1 and NHS-2). The latter contained the inhibitor, denominated γCdcPLI. The molecular mass of γCdcPLI, determined by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), was 22,340 Da. Partial sequences obtained by Edman degradation and by mass spectrometry (MALDI-TOF/TOF), showed similarity, as expected, to other related inhibitors. Circular dichroism (CD) analysis showed the presence of approximately 22% alpha helices and 29% beta sheets in the protein secondary structure. Additionally, CD studies also indicated no significant changes in the secondary structure of γCdcPLI when it is complexed to BpPLA2-TXI. On the other hand, dynamic light scattering (DLS) assays showed a temperature-dependent oligomerization behavior for this inhibitor. Biochemical analyses showed γCdcPLI was able to inhibit the enzymatic, cytotoxic and myotoxic activities of PLA2s. Structural and functional studies performed on this inhibitor may elucidate the action mechanisms of PLA2 inhibitors. In addition, we hope this study may contribute to investigating the potential use of these inhibitors for the treatment of snakebite or inflammatory diseases in which PLA2s may be involved.

The influence of agricultural and urban contamination on leech infestation of freshwater turtles, Phrynops geoffroanus, taken from two areas of the Uberabinha River.

Infestation by leeches on adult freshwater turtles Phrynops geoffroanus in two areas of the Uberabinha River, in Uberlândia, was investigated during the summer (January 2000). Fifty eight turtles were trapped: 26 (12 males, 14 females) in an agricultural area and 32 (16 males, 16 females) in an area with a more urban environment. The females present larger length and body mass (291.77+/-29.18 mm; 2233.30+/-511.40 g) than males (259.71+/-33.15 mm; 1488.68+/-529.35 g). Blood samples were drawn from the retrorbital sinus using heparinized micro-hematocrit capillary tubes, for microscopic and direct examination for intracellular parasites. Ectoparasitism by leeches, Placobdella bistriata, on young and adult animals was observed in the limb cavities of 28.1% of the urban area turtles. No leeches were found on the turtles in the agricultural area. Hemogregarine erythrocytic gametocytes were found in 15.4% of the agricultural area turtles, and in 37.5% of the urban area turtles. Concurrent parasitism by leeches and hemogregarines on the same individuals was seen on six turtles. Infections with microorganisms (bacteria and fungus) facilitated by the bites of leeches were not observed in another study on the same sample of infested turtles. However, 19% of the turtles collected in the agricultural area and 15% of the turtles from the urban area presented normocytic acute anemia. The occupation of the soil apparently interfered in the aquatic ecosystem favoring parasitism in the urban area.