Biomedical subjects

H S Selistre-de-Araujo

Publications and source records attributed to H S Selistre-de-Araujo.

17 recordsLinked to original sources

PubMed2006

The effect of the myotoxic Lys49 phospholipase A(2) from Agkistrodon contortrix laticinctus snake venom on Na+/K+ -ATPase activity of toad bladders.

ACLMT is a myotoxic Lys49 phospholipase A(2) isolated from the venom of the snake Agkistrodon contortrix laticinctus. We have previously shown that ACLMT increases baseline water transport and partially inhibits vasopressin-stimulated water transport across toad bladders due to an increase in cytosolic calcium. However, these evidences provide insufficient insight into the mechanisms involved in the effects of ACLMT on membrane permeability. In an attempt to better understand such mechanisms, the current study aimed to investigate whether the Na(+)/K(+)-ATPase activity of isolated toad bladders can be affected by the ACLMT and the synthetic peptide from its C-terminal region. The toxin significantly decreased the Na(+)/K(+)-ATPase, while the peptide did not alter it. These findings suggest that the effects of ACLMT on membrane permeability may be due to the inhibition of the Na(+)/K(+)-ATPase activity, and that the C-terminal region may not play a relevant role in this effect. This study contributes toward a better understanding of the mechanisms involved in the toxicity of the snake venom Lys49 PLA(2) myotoxins on biological tissues.

Agkistrodon↗

PubMed2006

Snake venom metalloproteases--structure and function of catalytic and disintegrin domains.

Snake venoms are relevant sources of toxins that have evolved towards the engineering of highly active compounds. In the last years, research efforts have produced great advance in their understanding and uses. Metalloproteases with disintegrin domains are among the most abundant toxins in many Viperidae snake venoms. This review will focus on the structure, function and possible applications of the metalloprotease and disintegrin domains.

Amino Acid Sequence↗

PubMed2005

Alternagin-C, a disintegrin-like protein from the venom of Bothrops alternatus, modulates alpha2beta1 integrin-mediated cell adhesion, migration and proliferation.

The alpha2beta1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, competitively interacts with the alpha2beta1 integrin, thereby inhibiting collagen binding. When immobilized in plate wells, ALT-C supports the adhesion of fibroblasts as well as of human vein endothelial cells (HUVEC) and does not detach cells previously bound to collagen I. ALT-C is a strong inducer of HUVEC proliferation in vitro. Gene expression analysis was done using an Affimetrix HU-95A probe array with probe sets of approximately 10,000 human genes. In human fibroblasts growing on collagen-coated plates, ALT-C up-regulates the expression of several growth factors including vascular endothelial growth factor, as well as some cell cycle control genes. Up-regulation of the vascular endothelial growth factor gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates protein kinase B phosphorylation, a signaling event involved in endothelial cell survival and angiogenesis. In human neutrophils, ALT-C has a potent chemotactic effect modulated by the intracellular signaling cascade characteristic of integrin-activated pathways. Thus, ALT-C acts as a survival factor, promoting adhesion, migration and endothelial cell proliferation after binding to alpha2beta1 integrin on the cell surface. The biological activities of ALT-C may be helpful as a therapeutic strategy in tissue regeneration as well as in the design of new therapeutic agents targeting alpha2beta1 integrin.

Animals↗

PubMed2004

Effects of a myotoxic Asp49 phospholipase A2 (ACL-I PLA2) isolated from Agkistrodon contortrix laticinctus snake venom on water transport in the isolated toad urinary bladder.

An Asp49 PLA2 (ACL-I PLA2) was purified from the venom of Agkistrodon contortrix laticinctus by gel filtration and cation-exchange chromatography. It has a relative molecular mass of 14,000, and its N-terminal sequence has more than 65% of identity with other snake venom PLA2s. ACL-I PLA2 injected into the Tibialis anterior muscle of rats and mice at doses of 0.3 and 1.6 mg/kg, respectively, induced muscle fiber necrosis, cellular infiltration and edema 3 and 48 h after injection. The effect of the purified enzyme on water permeability was tested in the isolated toad urinary bladder. Water flow through the membrane was measured gravimetrically in bag preparations of the bladder. ACL-I PLA2 (20 nM) did not significantly alter the water permeability in the bladder preparations, whereas ACL myotoxin (ACLMT), a Lys49 PLA2 isolated from the same venom, at similar concentration significantly increased (81%) the water permeability. However, both toxins inhibited the AVP-stimulated water permeability. These results strongly suggest that PLA2 activity is not involved in the ACLMT effect on water transport and the effect of ACL-I PLA2 myotoxin on membrane permeability is mediated by mechanisms that are different in comparison to ACLMT.

Agkistrodon↗

PubMed2004

The effect of post-translational modifications on the hemorrhagic activity of snake venom metalloproteinases.

Metalloproteinases (MPs) are Zn(+)-dependent endoproteolytic enzymes, abundant in crotalid and viperid snake venoms. Most snake venom metalloproteinases (svMPs) are active on extracellular matrix components and this effect is thought to result in bleeding as a consequence of the basement membrane disruption in capillaries. Jararhagin and ACLH are hemorrhagic svMPs from Bothrops jararaca and Agkistrodon contortrix laticinctus venom, respectively. Both enzymes demonstrate proteolytic activity on fibrinogen and fibronectin and jararhagin inhibits collagen-induced platelet aggregation in vitro. This work describes the expression, purification and successful refolding of the recombinant ACLH zymogen (rPRO-ACLH) as well as the catalytic domain of jararhagin (rCDJARA). The heterologous proteins were produced in E. coli, an in vivo expression system that does not make post-translational modifications. The recombinant refolded proteins did not show any hemorrhagic activity in mice skin, as well as the native deglycosylated jararhagin and ACLH. However, they preserved their proteolytic activity on fibrinogen and fibronectin. It seems that the hemorrhagic properties of these hemorrhagins are dependent on post-translational modifications, whereas their proteolytic activity is not dependent on such modifications.

Agkistrodon↗

PubMed2004

Effects of ACL myotoxin, a Lys49 phospholipase A(2) from Agkistrodon contortrix laticinctus snake venom, on water transport in the isolated toad urinary bladder.

ACL myotoxin (ACLMT) is a Lys49 phospholipase A(2)-like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus. The aim of this work was to study the effect of ACLMT on water transport in the toad bladder. Water flow through the membrane was measured gravimetrically in bag preparations of the bladder. ACLMT (20 nM) increased the baseline water flow and partially inhibited arginine-vasopressin (AVP), 8-chlorophenylthio-cAMP (8-CPT-cAMP) and forskolin-stimulated water flow. The effect of ACLMT on baseline or AVP-stimulated water flow was prevented by lanthanum (0.1 mM) indicating that the effect of ACLMT on water transport may be mediated through an increase in intracellular calcium. The effect of ACLMT on baseline water flow was also prevented by nifedipine (0.1 mM) indicating the participation of exogenous calcium in this effect. Carbachol (0.1 mM) has been shown to enhance baseline water flow while inhibiting AVP-stimulated water flow. The effects of ACLMT and carbachol on baseline water flow and AVP-stimulated water flow were not additive, suggesting that both agents alter water transport by a similar mechanism. Indomethacin (10 microM) reduced the effect of ACLMT on forskolin-stimulated water flow, suggesting an increase in prostaglandin biosynthesis. These results suggest that the effects of ACLMT on water transport may be mediated by increasing intracellular calcium and stimulation prostaglandin biosynthesis.

Agkistrodon↗

PubMed2004

Effect of a recombinant Lys49PLA2 myotoxin and Lys49PLA2-derived synthetic peptides from Agkistrodon species on membrane permeability to water.

The aim of this work was to study the effect of recombinant ACL myotoxin, a Lys49PLA2 from Agkistrodon contortrix laticinctus snake venom and Lys49PLA2-derived synthetic peptides corresponding to the region 115-129 of venom of the two different Agkistrodon species on water permeability in the toad urinary bladder. The water flow through the membrane was measured gravimetrically in bag preparations of the bladder. The addition of recombinant ACL myotoxin-MBP (maltose binding protein) fusion protein (10 nM) to the bathing solution significantly increased (above 60%) the water transport compared with the control hemibladders. The addition of the Lys49PLA2-derived synthetic peptides in several concentrations to the bathing solution did not affect the water transport across membrane. These results suggest that the ACL myotoxin effect on water transport is not related to the cytotoxic C-terminal region.

Agkistrodon↗

PubMed2004

Comparative analysis of the catalytic domain of hemorrhagic and non-hemorrhagic snake venom metallopeptidases using bioinformatic tools.

Snake venom metalloproteases (SVMPs) are a set of interesting enzymes that are one of the major components of venom affecting hemostasis. A great challenge since their discovery has been to find molecular features responsible for their hemorrhagic potency and many attempts have been made without any consistent result. Here we describe a series of comparisons between the catalytic domains of hemorrhagic and non-hemorrhagic SVMPs made with the help of bioinformatics. These involved sequence and structure-based multiple alignments, phylogenetic reconstruction, predicted physical and chemical properties, motif scanning and structural analyses. Although hemorrhagic activity seems to be complex, involving multiple factors, we found some molecular characteristics that may influence the toxic effects. Among these findings, it was possible to use a molecular surface feature to subdivide the P-I class in hemorrhagic and non-hemorrhagic SVMPs. It was also possible to suggest a role for the conserved Asp148 and Ser176 residues in the stabilization of the active site.

Animals↗

PubMed2003

BaG, a new dimeric metalloproteinase/disintegrin from the Bothrops alternatus snake venom that interacts with alpha5beta1 integrin.

The alpha(5)beta(1) integrin is one of the major fibronectin receptors which plays an essential role in the adhesion of normal and tumor cells to extracellular matrix. Here, we describe the isolation and characterization of a novel dimeric metalloproteinase/disintegrin, which is an inhibitor of fibronectin binding to the alpha(5)beta(1) integrin. This protein (BaG) was isolated from the venom of the South American snake Bothrops alternatus by gelatin-Sepharose affinity and anion exchange chromatography. The molecular mass of BaG was approximately 130 kDa under non-reducing conditions and 55 kDa under reducing conditions by SDS-PAGE. BaG shows proteolytic activity on casein that was inhibited by EDTA. 1,10-phenanthroline-treated BaG (BaG-I) inhibits ADP-induced platelet aggregation with an IC(50) of 190 nM. BaG-I inhibits fibronectin-mediated K562 cell adhesion with an IC(50) of 3.75 microM. K562 cells bind to BaG-I probably through interaction with alpha(5)beta(1) integrin, since anti-alpha(5)beta(1) antibodies inhibited K562 cell adhesion to BaG-I. In addition, BaG-I induces the detachment of K562 cells that were bound to fibronectin. In summary, we have purified a novel, dimeric snake venom metalloproteinase/disintegrin that binds to the alpha(5)beta(1) integrin.

Amino Acid Sequence↗

PubMed2003

Cell adhesion and cytotoxicity studies over polyanionic collagen surfaces with variable negative charge and wettability.

This work describes the cytotoxicity, and the cell adhesion behavior of K562 cell line from human erythroleukemia transfected with the DNA for the alpha(2)beta(1) integrin over type-I collagen matrices with variable degree of carboxyl group and wettability. The results showed that type-I collagen materials with variable degree of carboxyl group prepared by selective hydrolysis of carboxyamide side chains of Asn and Gln residues present in the protein, independently from the extent of side chain hydrolysis, was characterized by preserved triple helix structure for materials with a carboxyl group content up to 87 +/- 17. Imbibition and wettability increased linearly with increasing carboxyl group content from 46 +/- 12 to 87 +/- 17, and no signs of cytotoxicity were detected. Nevertheless, in comparison to native collagen, K562 cell adhesion to PACMs was significantly improved by factors ranging from 1.60 to 1.47x, with the reduction in cell adhesion observed with increasing carboxyl content attributed to a balance between the inhibition of increasing negative charge and the stimulation by increased wettability. On the other hand, the overall improvement of K562 cell adhesion to polyanionic collagen was attributed to the introduction of new distinct motifs described as the minimal active recognition sequence for alpha(2)beta(1) integrins binding with type-I collagen produced as a result of Asn-Gly Glu-Ala alpha2(I)294-297, and Gly Gln-Arg-Gly Val-Val carboxyamide side chains hydrolysis.

Animals↗

PubMed2001

Crystallization and preliminary X-ray analysis of jararhagin, a metalloproteinase/disintegrin from Bothrops jararaca snake venom.

Jararhagin is a toxic protein, isolated from the venom of the snake Bothrops jararaca, which is composed of a metalloprotease domain coupled to a disintegrin/cysteine-rich domain. It induces local haemorrhage owing to the proteolytic digestion of the basement membrane of capillaries. Jararhagin also cleaves the alpha(2)beta(1) integrin on the surface of platelets, thereby acting as a potent inhibitor of collagen-induced platelet aggregation. Crystals of jararhagin were obtained by the vapour-diffusion technique at 273 K in 200 mM sodium acetate, 100 mM cacodylate buffer pH 6.5 and 30% PEG 8000. Diffraction data have been obtained to a resolution of 2.8 A from a single frozen crystal, which belonged to space group P2(1)2(1)2(1) with unit-cell parameters a = 73.7, b = 100.3, c = 133.4 A. The asymmetric unit contains two jararhagin molecules and has a solvent content of 45%. A molecular-replacement solution has been obtained using a homology-built model based on the crystal structure of acutolysin, a haemorrhagic zinc metalloproteinase from the venom of the snake Agkistrodon acutus; attempts are under way to locate the remaining domains.

Animals↗

PubMed2001

Expression of an active recombinant lysine 49 phospholipase A(2) myotoxin as a fusion protein in bacteria.

ACL myotoxin (ACLMT) is a K49 phospholipase A(2)-like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus (broad-banded copperhead) that induces necrosis of skeletal muscle. We have previously cloned and sequenced the cDNA coding for ACLMT from a venom gland cDNA library. In order to perform structure and function studies, we have developed an expression system for production of ACLMT as a fusion protein with maltose binding protein (MBP) from the periplasm of bacteria, using the pMAL-p2 expression vector. The cDNA coding for the mature toxin without the signal peptide was amplified by PCR and subcloned into the pMAL-p2 vector. The new plasmid (pMAL-MT) was used to transform BL21(DE3) E. coli cells. Culture of transformed cells induced with IPTG led to the expression of a 60 kDa fusion protein which strongly reacts with anti-native ACLMT antibodies. The fusion protein was purified from the bacterial periplasm by affinity chromatography in an amylose column and by gel filtration. The purified fusion protein (MBP-rACLMT) was able to induce necrosis of skeletal muscle of mice very similar to that caused by the native myotoxin.

ATP-Binding Cassette Transporters↗

PubMed2001

Spectral characteristics of a compound altering cytochrome P450 spectra from vertebrate microsomes suggest that it is a functional protein.

A peak near 420 nm interfering with the spectral detection of cytochrome P450 has been reported for invertebrates and fish. It has been variously suggested to be a breakdown product of P450, or a hemoprotein with unknown functions. Similar spectra were observed in the present work with a neotropical fish, an amphibian, and rodents. Comparative analysis showed that difference spectra resulted from an unknown hemoprotein and neither from P420, nor from hemoglobin, that may contaminate animal microsomes. Seasonal appearance of this protein was observed and its spectrum described. This protein completely substituted P450 in spectra of liver microsomes of fish and rodents collected in the summer, while in the winter the same animals displayed either the classic P450 spectra (rodents) or those accompanied with the low-intensity 421-nm peak (fish). We suggest that the compound visualized in P450 spectra is a functional protein and not an artifact. The possibility that an unknown protein may substitute for cytochrome P450 in microsomes under certain environmental conditions and play a role in animal adaptation to unfavorable environmental fluctuations is discussed.

Animals↗

PubMed2000

The disintegrin-like domain of the snake venom metalloprotease alternagin inhibits alpha2beta1 integrin-mediated cell adhesion.

The alpha2beta1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Here we describe the isolation of a novel metalloproteinase/disintegrin, which is a potent inhibitor of the collagen binding to alpha2beta1 integrin. This 55-kDa protein (alternagin) and its disintegrin domain (alternagin-C) were isolated from Bothrops alternatus snake venom. Amino acid sequencing of alternagin-C revealed the disintegrin structure. Alternagin and alternagin-C inhibit collagen I-mediated adhesion of K562-alpha2beta1-transfected cells. The IC50 was 134 and 100 nM for alternagin and alternagin-C, respectively. Neither protein interfered with the adhesion of cells expressing alphaIIbeta3, alpha1beta1, alpha5beta1, alpha4beta1 alphavbeta3, and alpha9beta1 integrins to other ligands such as fibrinogen, fibronectin, and collagen IV. Alternagin and alternagin-C also mediated the adhesion of the K562-alpha2beta1-transfected cells. Our results show that the disintegrin-like domain of alternagin is responsible for its ability to inhibit collagen binding to alpha2beta1 integrin.

Amino Acid Sequence↗

PubMed2000

Expression, refolding, and activity of a recombinant nonhemorrhagic snake venom metalloprotease.

Snake venoms are rich sources of proteases that strongly affect the vascular system, by promoting blood coagulation, hemorrhage, and fibrinolysis. Hemorrhagic activity is mostly due to the enzymatic action of metalloproteases on capillary basement membrane components, such as collagen IV, laminin, and fibronectin. A few low-molecular-weight snake venom metalloproteases (svMP) have been described as being devoid of hemorrhagic activity, but they have strong direct-acting fibrinolytic activity that could be very helpful in thrombosis therapy. We have developed an expression system for production of a recombinant svMP from a cDNA (ACLPREF) coding for a small metalloprotease (ACLF) with three disulfide bonds from an Agkistrodon contortrix laticinctus (broad-banded copperhead) venom gland cDNA library. The mature protein-coding region was amplified by PCR and subcloned into the pET28a vector, and the resulting plasmid was used to transform BL21(DE3) Escherichia coli cells. Culture of the transformants at either 37 or 20 degrees C led to the overexpression of an insoluble and inactive 30-kDa protein after 1.0 mM IPTG induction. The expressed protein (rACLF) was recovered from inclusion bodies with 6 M buffered urea solution and purified on a nickel-Sepharose column followed by gel filtration chromatography, both under denaturing conditions. After treatment with dithiothreitol, protein refolding was performed by gradual removal of the denaturing agent by dialysis. The refolded recombinant protein was active in fibrin-agarose plates. The purified protein achieved a conformation similar to that of the native enzyme as judged by circular dichroism analysis.

Agkistrodon↗

PubMed2000

Determination of the three-dimensional structure of toxins by protein crystallography.

Protein crystallography has significantly contributed to the development of many areas of biochemical research, particularly in the understanding of phenomena related to molecular recognition. Examples include the formation of enzyme-substrate complexes (and their subsequent catalysis), host cell invasion by viruses, antigen neutralization and peptide display by proteins of the immune system and many others. More recently, protein crystallography has also proved to be of great value in unraveling the molecular basis of many diseases as well as in the development of new drugs for their treatment. The X-ray diffraction technique in the elucidation of macromolecular structures is situated at the interface between the traditional research fields of biology, biochemistry, chemistry and physics where researchers are united by a common interest in the detailed understanding of macromolecule function and its relationship to three-dimensional structure. The purpose of this review is to describe, without resort to mathematical detail, all of the necessary steps for the complete determination of a three-dimensional structure by X-ray diffraction techniques. The basic procedures used for protein isolation and crystallization, crystallographic data collection and analysis and, finally, structure determination and refinement are all briefly reviewed. As such our efforts are not directed towards the specialist. Rather, it is our hope that the information presented will aid interested readers from other fields in the understanding of more specialized literature and who may wish to employ the information contained therein in the planning of their biological research. We hope that in so doing we will make clear both the power and limitations of the technique.

Animals↗

PubMed1999

Isolation and structural characterization of a cytotoxic L-amino acid oxidase from Agkistrodon contortrix laticinctus snake venom: preliminary crystallographic data.

We have purified a cytotoxic L-amino acid oxidase (LAO) from Agkistrodon contortrix laticinctus snake venom by means of Superdex-200 gel filtration, followed by phenyl-Sepharose CL-4B chromatography. The purified enzyme (ACL LAO) is a dimer on gel filtration, with a M(r) of 60,000 for the monomer as estimated by SDS-PAGE. LAO activity was tested against 15 amino acids, but only 9 were oxidized by the enzyme, suggesting that it presents some degree of specificity. ACL LAO has apoptosis-inducing activity in an HL-60 cell culture assay. After 24 h treatment with 25 micrograms/ml of ACL LAO, the typical DNA fragmentation pattern of apoptotic cells was observed on agarose gel electrophoresis. NMR analysis showed the presence of a flavin mononucleotide prosthetic group. To solve its 3-D structure, crystals of the purified protein were grown in 0.1 M Tris-HCl, pH 8.5, and 2 M (NH(4))(2)SO(4). Diffraction data collected to 3.5 A showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 A, c = 183.45 A. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase.

Agkistrodon↗