The PLA2 inhibitor from Crotalus durissus terrificus blood plasma (CNF) inhibits group III-PLA2 from honeybee venom.

Crotalus neutralizing factor (CNF) is an endogenous glycoprotein from Crotalus durissus terrificus snake blood that inhibits secretory phospholipases A2 (sPLA2) from the Viperid but not from Elapid venoms (subgroups IA and IIA, respectively). In the present study, we demonstrated that CNF can inhibit group III-PLA2 from bee venom by forming a stable enzyme-inhibitor complex. This finding opens up new possibilities for the potential use of CNF and/or CNF-based derivatives in the therapeutics of bee stings.

Mapping possible interaction sites for crotoxin in CNF, a gamma PLA2 inhibitor from Crotalus durissus terrificus rattle snake, using SPOT synthesis.

Phospholipases A2 (PLA2s) are main components of snake venoms. Several snake species possess endogenous PLA2 inhibitors in their circulating blood, which are generally known as sbPLIs (an acronym for snake blood phospholipase A2inhibitors). The sbPLIs are categorized in three classes (alpha, beta or gamma) depending on the existence of distinguishing protein domains in their structure. The Crotalus durrissus terrificus venom has a highly neurotoxic PLA2 - crotoxin (CTX) - in its composition and the self-protection of the snake is mainly ensured by a sbγPLI named CNF (standing for Crotalusneutralizing factor). In an attempt to find smaller molecules able to inhibit the catalytic activity of CTX, in the present study we used linear peptide arrays to identify CNF segments possibly involved in the interaction with the toxin. Five reacting segments were identified as possible interacting regions. The target peptides were synthesized and located in the in silico CNF structure. Although all of them are exposed to the solvent, high concentrations were needed to inhibit the PLA2 activity of the whole venom or CTX. Limitations of the methodology employed and particular characteristics of CTX inhibition by CNF are discussed.

Identification, description and structural analysis of beta phospholipase A2 inhibitors (sbßPLIs) from Latin American pit vipers indicate a binding site region for basic snake venom phospholipases A2.

Several snake species possess, in their circulating blood, endogenous PLA2 inhibitors (sbPLIs) with the primary function of natural protection against toxic enzymes from homologous and heterologous venoms. Among the three structural classes of sbPLIs - named α, ß, and γ - the ß class (sbßPLIs) is the least known with only four identified sequences, so far. The last class of inhibitors encompass molecules with leucine rich repeats (LRRs) motifs containing repeating amino acid segments. In the present study, we identified and characterized putative sbßPLIs from the liver and venom glands of six Latin American pit vipers belonging to Bothrops and Crotalus genera. The inhibitor from Crotalus durissus terrificus snakes (CdtsbßPLI) was chosen as a reference for the construction of the first in silico structural model for this class of inhibitors, using molecular modeling and molecular dynamics simulations. Detailed analyses of the electrostatic surface of the CdtsbßPLI model and protein-protein docking with crotoxin B from homologous venoms predict the interacting surface between these proteins.

Endogenous phospholipase A2 inhibitors in snakes: a brief overview.

The blood plasma of numerous snake species naturally comprises endogenous phospholipase A2 inhibitors, which primarily neutralize toxic phospholipases A2 that may eventually reach their circulation. This inhibitor type is generally known as snake blood phospholipase A2 inhibitors (sbPLIs). Most, if not all sbPLIs are oligomeric glycosylated proteins, although the carbohydrate moiety may not be essential for PLA2 inhibition in every case. The presently known sbPLIs belong to one of three structural classes - namely sbαPLI, sbßPLI or sbγPLI - depending on the presence of characteristic C-type lectin-like domains, leucine-rich repeats or three-finger motifs, respectively. Currently, the most numerous inhibitors described in the literature are sbαPLIs and sbγPLIs, whereas sbßPLIs are rare. When the target PLA2 is a Lys49 homolog or an Asp49 myotoxin, the sbPLI is denominated a myotoxin inhibitor protein (MIP). In this brief overview, the most relevant data on sbPLIs will be presented. Representative examples of sbαPLIs and sbγPLIs from two Old World - Gloydius brevicaudus and Malayopython reticulatus - and two New World - Bothrops alternatus and Crotalus durissus terrificus - snake species will be emphasized.

Endogenous phospholipase A2 inhibitors in snakes: a brief overview

The blood plasma of numerous snake species naturally comprises endogenous phospholipase A2 inhibitors, which primarily neutralize toxic phospholipases A2 that may eventually reach their circulation. This inhibitor type is generally known as snake blood phospholipase A2 inhibitors (sbPLIs). Most, if not all sbPLIs are oligomeric glycosylated proteins, although the carbohydrate moiety may not be essential for PLA2 inhibition in every case. The presently known sbPLIs belong to one of three structural classes namely sbPLI, sbPLI or sbPLI depending on the presence of characteristic C-type lectin-like domains, leucine-rich repeats or three-finger motifs, respectively. Currently, the most numerous inhibitors described in the literature are sbPLIs and sbPLIs, whereas sbPLIs are rare. When the target PLA2 is a Lys49 homolog or an Asp49 myotoxin, the sbPLI is denominated a myotoxin inhibitor protein (MIP). In this brief overview, the most relevant data on sbPLIs will be presented. Representative examples of sbPLIs and sbPLIs from two Old World Gloydius brevicaudus and Malayopython reticulatus and two New World Bothrops alternatus and Crotalus durissus terrificus snake species will be emphasized.

Endogenous phospholipase A2 inhibitors in snakes: a brief overview

The blood plasma of numerous snake species naturally comprises endogenous phospholipase A2 inhibitors, which primarily neutralize toxic phospholipases A2 that may eventually reach their circulation. This inhibitor type is generally known as snake blood phospholipase A2 inhibitors (sbPLIs). Most, if not all sbPLIs are oligomeric glycosylated proteins, although the carbohydrate moiety may not be essential for PLA2 inhibition in every case. The presently known sbPLIs belong to one of three structural classes - namely sbαPLI, sbβPLI or sbγPLI - depending on the presence of characteristic C-type lectin-like domains, leucine-rich repeats or three-finger motifs, respectively. Currently, the most numerous inhibitors described in the literature are sbαPLIs and sbγPLIs, whereas sbβPLIs are rare. When the target PLA2 is a Lys49 homolog or an Asp49 myotoxin, the sbPLI is denominated a myotoxin inhibitor protein (MIP). In this brief overview, the most relevant data on sbPLIs will be presented. Representative examples of sbαPLIs and sbγPLIs from two Old World - Gloydius brevicaudus and Malayopython reticulatus - and two New World - Bothrops alternatus and Crotalus durissus terrificus - snake species will be emphasized.(AU)

Immune response and protective efficacy of S9 ribosomal protein of Streptococcus pneumoniae in a model of sepsis.

Vaccination is the most promising strategy to reduce the incidence of pneumococcal infection. Although there are vaccines available, all of them are based on polysaccharide antigens (conjugated or not). In addition to their high cost, those vaccines do not cover all serotypes. To overcome these hindrances, we evaluated the immunogenicity and the protective efficacy of the S9 ribosomal protein of Streptococcus pneumoniae with the aim of developing a protein-based vaccine in the future. The gene encoding the S9 ribosomal protein was cloned in pET21-a expression vector, and the recombinant S9 protein was used to immunize mice. Significantly higher levels of anti-S9 immunoglobulin G were achieved (with predominance of immunoglobulin G1) in comparison with the control. Antibodies elicited against S. pneumoniae protein extract in rabbit recognized the recombinant S9 protein by Western blot, thus demonstrating its immunogenicity. Moreover, mice immunized with recombinant S9 protein and challenged with a virulent strain of S. pneumoniae presented a significant reduction of bacteremia after 24 h of infection as compared with the control. However, in the S9-immunized mice the onset of death was insignificantly delayed, but all of them died by the fourth day postinfection.

Evaluation of water-in-oil-in-water multiple emulsion and microemulsion as potential adjuvants for immunization with rabies antigen.

Water-in-oil-in-water (W/O/W) multiple emulsions and microemulsions have been studied as potential candidates to be formulated as adjuvants. In this work their application as adjuvants for rabies virus immunization was studied. The humoral response, the effective dose 50 and histology for the developed formulations were evaluated in mice and compared with those from traditional adjuvants. The microemulsion and W/O/W multiple emulsion adjuvants developed were able to induce humoral response in mice and the serum showed good in vivo protection. Compared to the other adjuvants evaluated, microemulsion was shown to be the best candidate for rabies immunization as it presented good potency against the virus and did not appear to cause any local reaction.

Leukotoxic activity of Actinobacillus Actinomycetemcomitans isolated from human and non-human primates PRIMATES

Actinobacillus actinomycetemcomitans is a clinically relevant periodontopathogenic Gram-negative coccobacillus that produces a leukotoxin of the RTX cytolysin family. In this study, we evaluated the leukotoxic activity of A. actinomycetemcomitans strains isolated from human and marmosets by Trypan blue exclusion and by the chemiluminescence assays. Among eight A. actinomycetemcomitans human strains studied, two (P2.17 and P8.12) were classified as high leukotoxin producers and among eight marmoset strains, one (M22.11) showed high leukotoxin production, as determined by Trypan blue exclusion assay. The reference strains ATCC 29523 and FDC Y4 respectively behaved like moderate and low producers. The chemiluminescence assay was used to evaluate the leukotoxic activity of M22.11 and P2.17 strains submitted to different growth conditions. Leukotoxic activity was detected on cells at the logarithmic phase and was similar under anaerobic and microaerophilic growth conditions. It was greatly reduced when cells were grown at glucose concentrations lower or higher than 0.75per cent (0.25per cent and 1.5per cent) in thioglycolate medium. Leukotoxin production mainly by the M22.11 strain was low in BHI broth, whereas production in TSB medium showed a similar level as in thioglycolate broth medium. Sodium bicarbonate at 10 mM did not affect leukotoxin production.