Biomedical subjects

S S Veiga

Publications and source records attributed to S S Veiga.

At least 19 recordsLinked to original sources

PubMed2005

Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom.

In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed jararaca). The proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography on CM Sepharose. The enzyme called leucurolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. The amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. The protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH2-terminus and containing no carbohydrate. The proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity=21.6 units/mg and 17.5 units/microg, respectively; crude venom=8.0 units/mg and 9.5 units/microg). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a cleaved the Ala14-Leu15 and Tyr16-Leu17 bonds in oxidized insulin B chain. The pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 microg) subcutaneously into mice.

Amino Acid Sequence↗

PubMed2004

Cytotoxic effect of a new 1,3,4-thiadiazolium mesoionic compound (MI-D) on cell lines of human melanoma.

The structural characteristics of mesoionic compounds, which contain distinct regions of positive and negative charges associated with a poly-heteroatomic system, enable them to cross cellular membranes and interact strongly with biomolecules. Potential biological applications have been described for mesoionic compounds. 1,3,4-Thiadiazolium mesoionic compound (MI-D), a new mesoionic compound, has been demonstrated to be extremely cytotoxic to B16-F10 murine melanoma cells when compared to fotemustine and dacarbazine, drugs of reference in melanoma treatment protocols, describing inhibition of tumours grown in vitro and in vivo. We now evaluate the effects of mesoionic compound MI-D on different human melanoma cell lines. The drug decreased the viability and proliferation of MEL-85, SK-MEL, A2058 and MEWO cell lines in vitro, showing a considerable cytotoxic activity on these human cells. Adhesion of MEL-85 cells was evaluated in the presence of the drug using different extracellular matrix (ECM) constituents. MI-D decreased MEL-85 adhesion to laminin, fibronectin and matrigel. The morphology and actin cytoskeleton organisation of MEL-85 cells were also modified on MI-D treatment. These results on human melanoma cell lines indicate that MI-D is a very encouraging drug against melanoma, a tumour that is extremely resistant to chemotherapy.

Cell Adhesion↗

PubMed2002

Morphological and biochemical evidence of blood vessel damage and fibrinogenolysis triggered by brown spider venom.

The venom of the brown spider is remarkable because it causes dermonecrotic injury, hemorrhagic problems, hemolysis, platelet aggregation and renal failure. The mechanism by which the venom causes hemorrhagic disorders is poorly understood. Rabbits intradermally exposed to the venom showed a local hemorrhage starting 1 h after inoculation and reaching maximum activity between 2 and 3 days. Biopsies examined by light and transmission electron microscopy showed subendothelial blebs, vacuoles and endothelial cell membrane degeneration in blood vessels, plasma exudation into connective tissue, and fibrin and thrombus formation within blood vessels. Loxosceles intermedia venom incubated with fibrinogen partially degrades Aalpha and Bbeta chains of intact fibrinogen, and significantly cleaves all Aalpha, Bbeta and gamma chains when they were separated or when fibrinogen is denatured by boiling. Proteolytic kinetic studies showed that the Aalpha chain is more susceptible to venom hydrolysis than the Bbeta chain. The fibrinogenolysis is blocked by ethylenediamine tetraacetic acid and 1,10-phenanthroline, but not by other protease inhibitors. Human plasma incubated with the venom had coagulation parameters such as prothrombin time, activated partial thromboplastin time and thrombin time increased. Through molecular sieve chromatography, we isolated a venom toxin of 30 kDa with fibrinogenolytic activity. We propose that the local and systemic hemorrhagic disorders evoked in loxoscelism are consequences of direct venom fibrinogenolysis together with cytotoxicity to subendothelial structures and endothelial cells in blood vessels.

Animals↗

PubMed2001

In vivo and in vitro cytotoxicity of brown spider venom for blood vessel endothelial cells.

The effect of brown spider (Loxosceles intermedia) venom on endothelial cells was investigated in vivo and in vitro. Morphological and ultrastructural observations by light microscopy and transmission electron microscopy showed that the venom acts in vivo upon vessel endothelial cells of rabbits that were intradermally injected, evoking vessel instability, cytoplasmic endothelial cell vacuolization, and blebs. Likewise, treatment of rabbit endothelial cells in culture with the venom led to loss of adhesion of the cells to the substrate. Endothelial cells in culture were metabolically radiolabeled with sodium [35S]-sulfate and the sulfated compounds (proteoglycans and sulfated proteins) from medium, cell surface, and extracellular matrix (ECM) were analyzed. Agarose gel electrophoresis and SDS-PAGE showed that the venom is active on the ECM and on cell surface proteoglycans, shedding these molecules into the culture medium. In addition, when purified heparan sulfate proteoglycan (HSPG) and purified laminin-entactin (LN/ET) complex were incubated with the venom we observed a partial degradation of the protein core of HSPG as well as the hydrolysis of entactin. The above results suggest that the L. intermedia venom has a deleterious effect on the endothelium of vessels both in vivo and in culture, removing important constituents such as HSPG and entactin that are involved in the adhesion of endothelial cells and of subendothelial ECM organization.

Animals↗

PubMed2001

A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins.

Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5)) and CHO-745 (2 x 10(5) and 5 x 10(5)) cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications.

Animals↗

PubMed2001

Extracellular matrix molecules as targets for brown spider venom toxins.

Loxoscelism, the term used to describe lesions and clinical manifestations induced by brown spider's venom (Loxosceles genus), has attracted much attention over the last years. Brown spider bites have been reported to cause a local and acute inflammatory reaction that may evolve to dermonecrosis (a hallmark of envenomation) and hemorrhage at the bite site, besides systemic manifestations such as thrombocytopenia, disseminated intravascular coagulation, hemolysis, and renal failure. The molecular mechanisms by which Loxosceles venoms induce injury are currently under investigation. In this review, we focused on the latest reports describing the biological and physiopathological aspects of loxoscelism, with reference mainly to the proteases recently described as metalloproteases and serine proteases, as well as on the proteolytic effects triggered by L. intermedia venom upon extracellular matrix constituents such as fibronectin, fibrinogen, entactin and heparan sulfate proteoglycan, besides the disruptive activity of the venom on Engelbreth-Holm-Swarm basement membranes. Degradation of these extracellular matrix molecules and the observed disruption of basement membranes could be related to deleterious activities of the venom such as loss of vessel and glomerular integrity and spreading of the venom toxins to underlying tissues.

Animals↗

PubMed2001

Heparan sulfate and control of cell division: adhesion and proliferation of mutant CHO-745 cells lacking xylosyl transferase.

We have examined the role of cell surface glycosaminoglycans in cell division: adhesion and proliferation of Chinese hamster ovary (CHO) cells. We used both wild-type (CHO-K1) cells and a mutant (CHO-745) which is deficient in the synthesis of proteoglycans due to lack of activity of xylosyl transferase. Using different amounts of wild-type and mutant cells, little adhesion was observed in the presence of laminin and type I collagen. However, when fibronectin or vitronectin was used as substrate, there was an enhancement in the adhesion of wild-type and mutant cells. Only CHO-K1 cells showed a time-dependent adhesion on type IV collagen. These results suggest that the two cell lines present different adhesive profiles. Several lines of experimental evidence suggest that heparan sulfate proteoglycans play a role in cell adhesion as positive modulators of cell proliferation and as key participants in the process of cell division. Proliferation and cell cycle assays clearly demonstrate that a decrease in the amount of glycosaminoglycans does not inhibit the proliferation of mutant CHO-745 cells when compared to the wild type CHO-K1, in agreement with the findings that both CHO-K1 and CHO-745 cells take 8 h to enter the S phase.

Animals↗

PubMed2000

Cellular prion protein binds laminin and mediates neuritogenesis.

Laminin (LN) plays a major role in neuronal differentiation, migration and survival. Here, we show that the cellular prion protein (PrPc) is a saturable, specific, high-affinity receptor for LN. The PrPc-LN interaction is involved in the neuritogenesis induced by NGF plus LN in the PC-12 cell line and the binding site resides in a carboxy-terminal decapeptide from the gamma-1 LN chain. Neuritogenesis induced by LN or its gamma-1-derived peptide in primary cultures from rat or either wild type or PrP null mice hippocampal neurons, indicated that PrPc is the main cellular receptor for that particular LN domain. These results point out to the importance of the PrPc-LN interaction for the neuronal plasticity mechanism.

Animals↗

PubMed2000

Structural and ultrastructural description of the venom gland of Loxosceles intermedia (brown spider).

The brown spider, genus Loxosceles, is becoming of great medical importance, with envenomation (Loxoscelism) occurring throughout the world. The biological activities of the brown spider venom usually include dermonecrotic lesions at the bite site accompanied by hemolytic and haemorrhagic effects and also by renal failure. The objective of the present study was to describe the histology of the venom gland of L. intermedia using glands from adult spiders which were investigated by light microscopy, using immunohistochemical and staining methods, by transmission electron microscopy, and by scanning electron microscopy. The organization of the venom gland of Loxosceles intermedia follows the general architecture of spiders' venom glands. Using light microscopy and transmission electron microscopy we observed that the venom glands of L. intermedia present two layers of striated muscle fibers, an external layer and an internal layer in touch with an extracellular matrix which is a basement membrane structure and a fibrillar collagen matrix separating the muscular region from epithelial cells of the venom gland. Muscle cells are multinucleated, with nuclei peripherally placed and their cytoplasm rich in sarcoplasmic reticulum, myofibrills and continuous Z lines. By using scanning electron microscopy we can detect muscular cells from external layer as branching cells. Epithelial cells have their cytosol extremely rich in rough endoplasmic reticulum, mitochondria collection, Golgi apparatus, interdigitating membranes and secretory vesicles that ultimately accumulate the venom, a complex protein mixture.

Animals↗

PubMed2000

Identification of high molecular weight serine-proteases in Loxosceles intermedia (brown spider) venom.

High molecular weight serine-proteases have been identified in Loxosceles intermedia (brown spider) venom. The mechanism by which Loxosceles spp venoms cause dermonecrotic injury (a hallmark of loxoscelism) is currently under investigation, but it seems to be molecularly complex and in some instance proteases might be expected to play a role in this skin lesion. In the present investigation, when we submitted L. intermedia venom to linear gradient 3-20% SDS-PAGE stained by a monochromatic silver method we detected a heterogeneous protein profile in molecular weight, ranging from 850- to 5-kDa. In an attempt to detect zymogen molecules of proteolytic enzymes, venom aliquots were treated with several exogenous proteases. Among them, trypsin activated two gelatinolytic molecules of 85- and 95-kDa in the venom. In experiments of hydrolysis inactivation using different protease inhibitors for four major class of proteases, we detected that only serine-type protease inhibitors were able to inactivate the 85- and 95-kDa enzymes in the venom. An examination of the 85- and 95-kDa gelatinolytic activities as a function of pH showed that these proteases had no apparent activities at pH below 5.0 and higher than 9.0 and displayed little activity at pH 6.0. with the optimal pH for their activities ranging from 7.0 to 8.0. Evaluation of the functional specificities of the 85- and 95-kDa venom proteases showed that these proteases efficiently degrade gelatin (denatured collagen) but have no proteolytic activity on hemoglobin, immunoglobulin, albumin, librinogen or laminin, suggesting specificity of their proteolytic actions. We describe here two serine-proteases activities in L. intermedia venom probably involved in the harmful effects of the venom.

Animals↗

PubMed2000

Effect of brown spider venom on basement membrane structures.

Loxoscelism or necrotic arachnidism are terms used to describe lesions and reactions induced by bites (envenomation) from spiders of the genus Loxosceles. Envenomation has been reported to provoke dermonecrosis and haemorrhage at the bite site and haemolysis, disseminated intravascular coagulation and renal failure. The purpose of this work was to study the effect of the venom of the brown spider Loxosceles intermedia on basement membrane structures and on its major constituent molecules. Light microscopy observations showed that L. intermedia venom obtained through electric shock, which reproduces two major signals of Loxoscelism in the laboratory, exhibits activity toward basement membrane structures in mouse Engelbreth-Holm-Swarm (EHS) sarcoma. Basement degradation was seen by a reduced periodic acid-Schiff (PAS) and alcian blue staining as well as by a reduced immunostaining for laminin when compared to control experiments. Electron microscopy studies confirmed the above results, showing the action of the venom on EHS-basement membranes and demonstrating that these tissue structures are susceptible to the venom. Using purified components of the basement membrane, we determined through SDS-PAGE and agarose gel that the venom is not active toward laminin or type IV collagen, but is capable of cleaving entactin and endothelial heparan sulphate proteoglycan. In addition, when EHS tissue was incubated with venom we detected a release of laminin into the supernatant, corroborating the occurrence of some basement membrane disruption. The venom-degrading effect on entactin was blocked by 1, 10-phenanthroline, but not by other protease inhibitors such as PMSF, NEM or pepstatin-A. By using light microscopy associated with PAS staining we were able to identify that 1,10-phenanthroline also inhibits EHS-basement membrane disruption evoked by venom, corroborating that a metalloprotease of venom is involved in these effects. Degradation of these extracellular matrix molecules and the observed susceptibility of the basement membrane could lead to loss of vessel and glomerular integrity, resulting in haemorrhage and renal problems after envenomation.

Animals↗

PubMed1999

Oligosaccharide residues of Loxosceles intermedia (brown spider) venom proteins: dependence on glycosylation for dermonecrotic activity.

Loxosceles spp. (brown spider) envenomation has been reported to provoke dermonecrosis and haemorrhage at the bite site (a hallmark of accidents) and, to a lesser extent, thrombocytopenia, hemolysis and disseminated intravascular coagulation in some cases. Using lectin-immunolabeling, lectin-affinity chromatography, glycosidase and proteinase K treatments we were able to identify several venom N-glycosylated proteins with high-mannose oligosaccharide structures, complex-type glycoconjugates such as fucosylated glycans, but no galactose or sialic acid residues as complex sugars or glycosaminoglycan residues. Working with enzymatically or chemically deglycosylated venom we found that platelet aggregation (thrombocytopenic activity) as well as the fibronectinolytic and fibrinogenolytic (haemorrhagic) effects of the venom were sugar-independent when compared to glycosylated venom. Nevertheless, zymograph analysis in co-polymerized gelatin gels showed that enzymatic N-deglycosylation of loxolysin-B, a high-mannose 32-35 kDa glycoprotein of the venom with gelatinolytic metalloproteinase activity, caused a reduction of approximately 2 kDa in its molecular weight and a reduction of the gelatinolytic effect to a residual activity of 28% when compared to the glycosylated molecule, indicating a post-translational glycosylation-dependent gelatinolytic effect. Analysis of the dermonecrotic effect of the chemically or enzymatically N-deglycosylated venom detected only residual activity when compared with the glycosylated control. Thus, the present report suggests that oligosaccharide moieties play a role in the destructive effects of brown spider venom and opens the possibility for a carbohydrate-based therapy.

Animals↗

PubMed1999

Presence of a laminin-binding chondroitin sulfate proteoglycan at the cell surface of a human melanoma cell Mel-85.

Working with Mel-85 (a human melanoma cell line), we have been able to detect a laminin-binding molecule with an apparent molecular mass of 100/110 kDa (Mel-85-LBM). Reduction with beta-mercaptoethanol decreases its molecular mass but does not affect its ability to bind laminin. This laminin interaction seems to be very specific since Mel-85-LBM binds laminin, but not fibronectin, vitronectin or type I collagen in affinity chromatography experiments. The molecule has a negative net charge at physiological pH and binds laminin in a divalent cation dependent way. Mel-85-LBM was metabolically radiolabeled with sodium [35S]-sulfate and chemical beta-elimination of purified Mel-85-LBM releases chondroitin sulfate chains. Mel-85-LBM is also sensitive to chondroitinase ABC digestion. These findings show that this molecule is a chondroitin sulfate proteoglycan. The location of this proteoglycan at the cell surface is evidenced by experiments using a polyclonal antiserum raised against purified Mel-85-LBM, that specifically reacts with just one molecule by western blotting among Mel-85 total cell extract as well as produces a positive signal by flow cytometry and a fluorescence profile of Mel-85 cells adhered on laminin.

Cell Adhesion↗

PubMed1998

Detection and characterization of metalloproteinases with gelatinolytic, fibronectinolytic and fibrinogenolytic activities in brown spider (Loxosceles intermedia) venom.

By studying Loxosceles intermedia (Brown spider) venom we were able to detect a proteolytic action on fibronectin and fibrinogen but an inability to degrade full length laminin, type I and type IV collagens. By studying enzyme inhibitors we observed that divalent metal chelators as EDTA and 1,10-phenanthroline completely blocked this cleaving action whereas serine-protease inhibitors, thiol-protease inhibitor and acid-protease inhibitor showed little or no effect on the proteolytic activity of the venom indicating involvement of a metalloproteinase. Zymogram analysis of venom detected a 35 kDa molecule with gelatinolytic activity. The metalloproteinase nature was further supported by its sensitivity to 4-aminophenyl mercuric acetate (APMA) treatment which decreased its molecular weight to 32 kDa, inhibition of its gelatinolytic effect by 1,10-phenanthroline and its elution from gelatin-sepharose affinity beads. In addition, zymogram experiments using fibronectin and fibrinogen as substrates detected a fibronectinolytic and fibrinogenolytic band at 28 kDa which changed its electrophoretic mobility to 20 kDa band after organomercurial treatment. The inhibitory effect of 1,10 phenanthroline and APMA sensitivity on this proteolytic effect confirmed the presence of a second metalloproteinase in the venom. The data presented herein describe two invertebrate metalloproteinases in L. intermedia venom with different specificities one gelatinolytic and another, fibronectinolytic and fibrinogenolytic, probably involved in the harmful effects of the venom.

Animals↗

PubMed1997

Post-translational modifications of alpha5beta1 integrin by glycosaminoglycan chains. The alpha5beta1 integrin is a facultative proteoglycan.

Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype alpha5beta1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified alpha5beta1 integrin as the only sulfated integrin among beta1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of alpha5beta1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl2, 8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical beta-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the alpha5beta1 heterodimer and alpha5 and beta1 integrin subunits were proteoglycans. The importance of alpha5beta1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.

Cell Adhesion↗

PubMed1997

Complementary hydropathy identifies a cellular prion protein receptor.

Prions, the etiological agents for infectious degenerative encephalopathies, act by entering the cell and inducing conformational changes in PrPC (a normal cell membrane sialoglycoprotein), which result in cell death. A specific cell-surface receptor to mediate PrPC and prion endocytosis has been predicted. Complementary hydropathy let us generate a hypothetical peptide mimicking the receptor binding site. Antibodies raised against this peptide stain the surface of mouse neurons and recognize a 66-kDa membrane protein that binds PrPC both in vitro and in vivo. Furthermore, both the complementary prion peptide and antiserum against it inhibit the toxicity of a prion-derived peptide toward neuronal cells in culture. Such reagents might therefore have therapeutic applications.

Amino Acid Sequence↗

PubMed1997

Immunohistochemical and biochemical analysis of laminin in neonatal rat first molars.

Rabbit polyclonal antibody against mouse EHS laminin was used to investigate the distribution and composition of laminin in the rat first molar tooth germ. Immunohistochemical analysis showed that laminin is expressed in the inner and outer epithelia of the enamel organ and in small blood vessels in the dental papilla and strellate reticulum. Immunoblots revealed that tooth germ laminin differs from EHS laminin. Tooth germ laminin contains beta chains, while the alpha 1 chain is substituted by a 300-kDa chain. Two-dimensional electrophoresis analysis of tooth germ extract showed that beta chains appeared as four spots with approximate pI values of 6.6, 7.5, 7.8 and 8.5. These results indicate that more than-one type of laminin isoform is present in the first molar tooth germ. Additionally, we have shown that despite the early degradation of tooth germ basement membrane, the laminin molecule is still intact at the time of birth.

Animals↗

PubMed1996

Detection of post-translational sulfation of alpha-5 beta-1 integrin and its role in integrin-fibronectin binding.

Fibronectins are glycoproteins of the extracellular matrix composed of two 220-kDa polypeptide chains named A and B bound by two disulfide bridges. Both chains when digested with proteolytic enzymes give rise to six different domains named I to VI that are involved in the ligand properties of this molecule. Fibronectins bind fibrin, collagen, glycosaminoglycan residues and several integrins. In this study, using metabolic radiolabeling of alpha 5 beta 1 integrin with sodium sulfate, an immunoprecipitation reaction, inhibition of sulfate incorporation and a fibronectin-binding assay, we were able to detect this integrin as a sulfated molecule and this sulfation appears to regulate the integrin-fibronectin binding.

Collagen↗