Effect of the synthetic coumarin, ethyl 2-oxo-2H-chromene-3-carboxylate, on activity of Crotalus durissus ruruima sPLA2 as well as on edema and platelet aggregation induced by this factor.

We show that ethyl 2-oxo-2H-chromene-3-carboxylate (EOCC), a synthetic coumarin, irreversibly inhibits phospholipase A(2) (sPLA2) from Crotalus durissus ruruima venom (sPLA2r) with an IC(50) of 3.1 +/- 0.06 nmol. EOCC strongly decreased the V(max) and K(m), and it virtually abolished the enzyme activity of sPLA2r as well as sPLA2s from other sources. The edema induced by sPLA2r + EOCC was less than that induced by sPLA2r treated with p-bromophenacyl bromide, which was more efficient at neutralizing the platelet aggregation activity of native sPLA2r. Native sPLA2r induced platelet aggregation of 91.54 +/- 9.3%, and sPLA2r + EOCC induced a platelet aggregation of 18.56 +/- 6.5%. EOCC treatment also decreased the myotoxic effect of sPLA2r. Mass spectrometry showed that EOCC formed a stable complex with sPLA2r, which increased the mass of native sPLA2r from 14,299.34 Da to 14,736.22 Da. Moreover, the formation of this complex appeared to be involved in the loss of sPLA2r activity. Our results strongly suggest that EOCC can be used as a pharmacological agent against the sPLA2 in Crotalus durissus sp. venom as well as other sPLA2s.

Purification and biological effects of a C-type lectin isolated from bothrops moojeni

Snake venom proteins from the C-type lectin family have very distinct biological activities despite their highly conserved primary structure, which is homologous to the carbohydrate recognition region of true C-type lectins. We purified a lectin-like protein (BmLec) from Bothrops moojeni venom and investigated its effect on platelet aggregation, insulin secretion, antibacterial activity, and isolated kidney cells. The BmLec was purified using two chromatographic steps: affinity chromatography and reverse phase high performance liquid chromatography (HPLC). BmLec showed a dose-dependent platelet aggregation and significantly decreased the bacterial growth rate in approximately 15 percent. During scanning electron microscopy, the profile of Xanthomonas axonopodis pv. passiflorae treated with lectin disclosed a high vesiculation and membrane rupture. BmLec induced a strong and significant increase in insulin secretion at 2.8 and 16.7 mM glucose concentrations, and this effect was seen in the presence of EGTA in both experiments. BmLec (10 µg/mL) increased the perfusion pressure, renal vascular resistance and urinary flow. The glomerular filtration rate and percentages of sodium, potassium and chloride tubular transport were reduced at 60 minutes of perfusion. Renal alterations caused by BmLec were completely inhibited by indomethacin in all evaluated parameters. In conclusion, the C-type lectin isolated from Bothrops moojeni affected platelet aggregation, insulin secretion, antibacterial activity and isolated kidney function.

Biological and structural characterization of a new PLA2 from the Crotalus durissus collilineatus venom.

In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A2 (PLA2) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80% with crotalic PLA2s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50% if compared to other sources of PLA2s such as from the Bothrops snake venom. Also, this PLA2 induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and p-BPB showed a similar reduction. The p-BPB did not reduce significantly the myotoxic activity induced by the PLA2, but the anhydrous acetic acid treatment and the pre-incubation of PLA2 with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiforae (Gram-negative), which was drastically reduced by incubation of this PLA2 with p-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or beta-wing region may be involved in the binding of this PLA2 to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein.

Structural and functional characterization of basic PLA2 isolated from Crotalus durissus terrificus venom.

The venom of Crotalus durissus terrificus was fractionated by reverse-phase HPLC to obtain crotapotins (F5 and F7) and PLA2 (F15, F16, and F17) of high purity. The phospholipases A2 (PLA2S) and crotapotins showed antimicrobial activity against Xanthomonas axonopodis pv. passiflorae, although the unseparated crotoxin did not. The F17 of the PLA2 also revealed significant anticoagulant activity, althrough for this to occur the presence of Glu 53 and Trp 61 is important. The F17 of the PLA2 showed allosteric behavior in the presence of a synthetic substrate. The amino acid sequence of this PLA2 isoform, determined by automatic sequencing, was HLLQFNKMLKFETRK NAVPFYAFGCYCGWGGQRRPKDATDRCCFVHDCCYEKVTKCNTKWDFYRYSLKSGY ITCGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPDSRCREPSETC. Analysis showed that the sequence of this PLA2 isoform differed slightly from the amino acid sequence of the basic crotoxin subunit reported in the literature. The homology with other crotalid PLA2 cited in the literature varied from 60% to 90%. The pL was estimated to be 8.15, and the calculated molecular weight was 14664.14 as determined by Tricine SDS-PAGE, two-dimensional electrophoresis, and MALDI-TOFF. These results also suggested that the enzymatic activity plays an important role in the bactericidal effect of the F17 PLA2 as well as that of anticoagulation, although other regions of the molecule may also be involved in this biological activity.

Cytotoxin production in phytopathogenic and entomopathogenic Serratia marcescens.

In this work, culture filtrates of entomopathogenic and phytopathogenic Serratia marcescens strains induced cytotoxic effects on CHO, Vero and HEp-2 cell lines. Morphological changes on sensitive cells were characterized by cell rounding and detachment as soon as 30 min of incubation, culminating in cell death after 24 h. The cytotoxic effect was completely neutralized by specific antiserum indicating that occur antigenic similarity among cytotoxins produced by these strains. The toxicity assays on plants showed that the culture supernatants did not provoke any visible morphological change and did not affect their growth. By contrast, the plants treated with bacterial suspension showed disease symptom, such as shriveling and decay of stores bulbus in onion and lettuce plantlets. In conclusion, this study show that phytopathogenic and entomopathogenic S. marcescens may produce a cytototoxin similar to that produced by clinical isolates and it is toxic to different mammalian cell lines. These results are especially important for studies involving this bacterium as biological control agent.

Detection of snail hemocyanin in the whole hemolymph by serologically specific electron microscopy.

This study demonstrates the applicability of the serologically specific electron microscopy (SSEM) technique in the detection of hemocyanin molecules in the whole hemolymph of the snail, Megalobulimulus ovatus. The results are positive and easily reproducible. The SSEM might be useful as a technique for taxonomic studies of snails as well as to study structural aspects of their hemocyanin molecules.