- Email: [email protected]
190. First Draft of the Genomic Organization of a PIII-SVMP Gene
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248 4 University Presbiteriana Mackenzie, Center of Biological and Health Sciences (CCBS), São Paulo, São Paulo, Brazil E-mail address: [email protected] (V.C.G. Soares).
Background: Glycation, a chemical modification of proteins with reducing sugars, indicating a possible explanation for the association between hyperglycemia and the wide variety of tissue pathologies. A good example is glycosiled hemoglobin that can be used as a molecular tool to characterize the severity of diabetes. In this in vitro study, we investigated the effect of glucose, galactose and lactose on the structure and function of BthTX-I, which was chosen as the structural model of sPLA2. Glucose is known and well characterized as one of the most important glycacanting agent of protein. Galactose is another monosaccharide with the same functional groups found on glucose and lactose which is the dimer of galactose and glucose were also evaluated in this study. Methods: The degree of glycosylation of BthTX-I with the various glycating agents was performed using affinity chromatography phenylboronate, which allowed the identification of glucose and galactose as two main glycants agents and purified the glycated BthTX-I form from the not. Results: The analysis in reverse-phase HPLC showed that both glucose and galactose significantly reduced hydrophobicity nature of BthTx-I. Moreover, both glucose and galactose significantly changed the spectrum of circular dichroism and intrinsic tryptophan fluorescence of BthTX-I. In addition, glucose and galactose also significantly reduced edema, myotoxicity and nephrotoxicity induced by native BthTX-I. Discussion: These results indicate the susceptibility of sPLA2 BthTx-I as non-enzymatic glycation by various sugars. It also describes the effects of glycation on the structure and pharmacological activity of BthTX-I. Conclusion: Moreover, as BthTX-I is an example of sPLA2 both in terms of structural and functional is possible to conclude that nonenzymatic glycation might be affect other types of secretory PLA2 in diabetes for example. Keywords: sPLA2, glycation, edema, myonecrosis. 10.1016/j.toxicon.2012.04.190
190. First Draft of the Genomic Organization of a PIII-SVMP Gene Libia Sanz 1, Robert A. Harrison 2, Juan J. Calvete 1
193
organization of genes coding for disintegrins, including loss of introns and coding regions, has been documented. However, details on the mechanisms of recruitment and molecular events underlying the origin and evolution of the SVMP multigene family remain elusive. Moreover, the genomic structure of any SVMP gene has not been reported, and thus the changes occurring after recruitment of the ancestral gene into the venom gland remain hidden in the snake genomes. Here we examine the gene structure of the 15652 bp Echis ocellatus pre-pro-EOC00089-like PIIISVMP gene asssembled from PCR-amplified sequences of overlapping genomic fragments. The gene comprises 12 exons interrupted by 11 introns. In a homology model of the EOC00089-like protein, the insertion of introns interrupting coding regions lie between secondary structure elements. LINEs L2/CR1 and RTE/BovB, SINE/Sauria, and a hobo-activator DNA transposon were identified within introns 1, 3, 7 and 8. Pairwise amino acid sequence comparisons between EOC00089-like PIII-SVMP and its closest orthologs, human and A. carolinensis ADAM28 showed that their ORFs share 42%/59%, 49%/69%, and 48%/65% (identity/similarity), respectively. The protein-coding positions interrupted by each of the 11 introns of the Echis PIII-SVMP gene are entirely conserved in the A. carolinensis and human ADAM28 genes. However, lizard and human ADAM28 genes contain 5 introns not present in E. ocellatus. Strikingly, none of the 11 introns shared between EOC00089-like PIII-SVMP and A. carolinensis ADAM28 genes exhibit conservation in size. Furthermore, Echis and Anolis introns exhibit quantitatively and qualitatively distinctions in their inserted retroelements. Our finding that retroelements are exclusively located within introns is consistent with the view that introns are added to genes presumably as transposable elements, and identifies introns as possible key elements in the recruitment and amplification process of SVMPs into the venom gland of extant snakes. Reptile genome sequencing projects may shed light on this aspect of the emergence and evolution of venom toxins. Furthermore, the organization of the PIII-SVMP reported here provides a genomic explanation for the emergence of dimeric disintegrin subunits encoded by short messengers. Keywords: gene organization, SVMP, intronic retroelements 10.1016/j.toxicon.2012.04.191
191. Dexamethasone Antagonizes the Myotoxic and Inflammatory Effect of Bothrops Venoms
1
Consejo Superior de Investigaciones Científicas, Valencia, Spain Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool, UK E-mail address: [email protected] (J.J. Calvete). 2
Review: The evolutionary pathway of snake venom toxins remains poorly understood. The origin of SVMPs has been inferred to have occurred following the split of the Pareatidae from the remaining Caenophidians, approximately 54-64 Mya, through recruitment, duplication, and neofunctionalization by positive Darwinian selection of a closely related body cellular ADAM 7 or 28 ancestor gene. The evolutionary history of viperid SVMPs is repeatedly punctuated by domain loss, and minimization of the
Fernando C. Patrão-Neto, Marcelo A. Tomaz, Marcos M. Machado, José Roberto Da S. Rocha-Junior, Paulo A. Melo Lab. Farmacologia das Toxinas, ICB, CCS, UFRJ, Rio de Janeiro, RJ Brazil E-mail address: [email protected] (P.A. Melo).
Background: We investigated the toxic activities from Bothrops genus snake venom using in vivo and in vitro experimental protocols in mice muscle and tested the protector effect of dexamethasone (DEXA) in different conditions, comparing it with the polyvalent antivenom. We also expanded the investigations on the antiophidic effect of the Eclipta prostrata (EP) crude extract.
Background: Glycation, a chemical modification of proteins with reducing sugars, indicating a possible explanation for the association between hyperglycemia and the wide variety of tissue pathologies. A good example is glycosiled hemoglobin that can be used as a molecular tool to characterize the severity of diabetes. In this in vitro study, we investigated the effect of glucose, galactose and lactose on the structure and function of BthTX-I, which was chosen as the structural model of sPLA2. Glucose is known and well characterized as one of the most important glycacanting agent of protein. Galactose is another monosaccharide with the same functional groups found on glucose and lactose which is the dimer of galactose and glucose were also evaluated in this study. Methods: The degree of glycosylation of BthTX-I with the various glycating agents was performed using affinity chromatography phenylboronate, which allowed the identification of glucose and galactose as two main glycants agents and purified the glycated BthTX-I form from the not. Results: The analysis in reverse-phase HPLC showed that both glucose and galactose significantly reduced hydrophobicity nature of BthTx-I. Moreover, both glucose and galactose significantly changed the spectrum of circular dichroism and intrinsic tryptophan fluorescence of BthTX-I. In addition, glucose and galactose also significantly reduced edema, myotoxicity and nephrotoxicity induced by native BthTX-I. Discussion: These results indicate the susceptibility of sPLA2 BthTx-I as non-enzymatic glycation by various sugars. It also describes the effects of glycation on the structure and pharmacological activity of BthTX-I. Conclusion: Moreover, as BthTX-I is an example of sPLA2 both in terms of structural and functional is possible to conclude that nonenzymatic glycation might be affect other types of secretory PLA2 in diabetes for example. Keywords: sPLA2, glycation, edema, myonecrosis. 10.1016/j.toxicon.2012.04.190
190. First Draft of the Genomic Organization of a PIII-SVMP Gene Libia Sanz 1, Robert A. Harrison 2, Juan J. Calvete 1
193
organization of genes coding for disintegrins, including loss of introns and coding regions, has been documented. However, details on the mechanisms of recruitment and molecular events underlying the origin and evolution of the SVMP multigene family remain elusive. Moreover, the genomic structure of any SVMP gene has not been reported, and thus the changes occurring after recruitment of the ancestral gene into the venom gland remain hidden in the snake genomes. Here we examine the gene structure of the 15652 bp Echis ocellatus pre-pro-EOC00089-like PIIISVMP gene asssembled from PCR-amplified sequences of overlapping genomic fragments. The gene comprises 12 exons interrupted by 11 introns. In a homology model of the EOC00089-like protein, the insertion of introns interrupting coding regions lie between secondary structure elements. LINEs L2/CR1 and RTE/BovB, SINE/Sauria, and a hobo-activator DNA transposon were identified within introns 1, 3, 7 and 8. Pairwise amino acid sequence comparisons between EOC00089-like PIII-SVMP and its closest orthologs, human and A. carolinensis ADAM28 showed that their ORFs share 42%/59%, 49%/69%, and 48%/65% (identity/similarity), respectively. The protein-coding positions interrupted by each of the 11 introns of the Echis PIII-SVMP gene are entirely conserved in the A. carolinensis and human ADAM28 genes. However, lizard and human ADAM28 genes contain 5 introns not present in E. ocellatus. Strikingly, none of the 11 introns shared between EOC00089-like PIII-SVMP and A. carolinensis ADAM28 genes exhibit conservation in size. Furthermore, Echis and Anolis introns exhibit quantitatively and qualitatively distinctions in their inserted retroelements. Our finding that retroelements are exclusively located within introns is consistent with the view that introns are added to genes presumably as transposable elements, and identifies introns as possible key elements in the recruitment and amplification process of SVMPs into the venom gland of extant snakes. Reptile genome sequencing projects may shed light on this aspect of the emergence and evolution of venom toxins. Furthermore, the organization of the PIII-SVMP reported here provides a genomic explanation for the emergence of dimeric disintegrin subunits encoded by short messengers. Keywords: gene organization, SVMP, intronic retroelements 10.1016/j.toxicon.2012.04.191
191. Dexamethasone Antagonizes the Myotoxic and Inflammatory Effect of Bothrops Venoms
1
Consejo Superior de Investigaciones Científicas, Valencia, Spain Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool, UK E-mail address: [email protected] (J.J. Calvete). 2
Review: The evolutionary pathway of snake venom toxins remains poorly understood. The origin of SVMPs has been inferred to have occurred following the split of the Pareatidae from the remaining Caenophidians, approximately 54-64 Mya, through recruitment, duplication, and neofunctionalization by positive Darwinian selection of a closely related body cellular ADAM 7 or 28 ancestor gene. The evolutionary history of viperid SVMPs is repeatedly punctuated by domain loss, and minimization of the
Fernando C. Patrão-Neto, Marcelo A. Tomaz, Marcos M. Machado, José Roberto Da S. Rocha-Junior, Paulo A. Melo Lab. Farmacologia das Toxinas, ICB, CCS, UFRJ, Rio de Janeiro, RJ Brazil E-mail address: [email protected] (P.A. Melo).
Background: We investigated the toxic activities from Bothrops genus snake venom using in vivo and in vitro experimental protocols in mice muscle and tested the protector effect of dexamethasone (DEXA) in different conditions, comparing it with the polyvalent antivenom. We also expanded the investigations on the antiophidic effect of the Eclipta prostrata (EP) crude extract.