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Francis S Markland - One of the best experts on this subject based on the ideXlab platform.

  • structure of acostatin a dimeric disintegrin from southern copperhead Agkistrodon Contortrix Contortrix at 1 7 a resolution
    Acta Crystallographica Section D-biological Crystallography, 2008
    Co-Authors: Natalia Moiseeva, Francis S Markland, Robert Bau, Stephen Swenson, Jun Yong Choe, Zhijie Liu, Marc Allaire
    Abstract:

    Disintegrins are a family of small (4–14 kDa) proteins that bind to another class of proteins, integrins. Therefore, as integrin inhibitors, they can be exploited as anticancer and antiplatelet agents. Acostatin, an αβ heterodimeric disintegrin, has been isolated from the venom of Southern copperhead (Agkistrodon Contortrix Contortrix). The three-dimensional structure of acostatin has been determined by macromolecular crystallography using the molecular-replacement method. The asymmetric unit of the acostatin crystals consists of two heterodimers. The structure has been refined to an R_(work) and R_(free) of 18.6% and 21.5%, respectively, using all data in the 20–1.7 A resolution range. The structure of all subunits is similar and is well ordered into N-terminal and C-terminal clusters with four intramolecular disulfide bonds. The overall fold consists of short β-sheets, each of which is formed by a pair of antiparallel β-strands connected by β-turns and flexible loops of different lengths. Conformational flexibility is found in the RGD loops and in the C-terminal segment. The interaction of two N-terminal clusters via two intermolecular disulfide bridges anchors the αβ chains of the acostatin dimers. The C-terminal clusters of the heterodimer project in opposite directions and form a larger angle between them in comparison with other dimeric disintegrins. Extensive interactions are observed between two heterodimers, revealing an αββα acostatin tetramer. Further experiments are required to identify whether the αββα acostatin complex plays a functional role in vivo.

  • Anti-Angiogenic Activity of Contortrostatin, a Disintegrin from Agkistrodon Contortrix Contortrix Snake Venom
    Angiogenesis, 2003
    Co-Authors: Vladislav Golubkov, Debra Hawes, Francis S Markland
    Abstract:

    Previous work in our laboratory has shown that contortrostatin (CN), a disintegrin from southern copperhead snake venom, possess anti-angiogenic activity. In the present study we further examined the anti-angiogenic activity of CN, focusing on the mechanisms of CN inhibition of angiogenesis. CN inhibited migration and invasion, and significantly altered Matrigel-induced tube formation of human umbilical vein endothelial cells (HUVEC), but did not affect cell viability, or MMP-2 and MMP-9 activity. Immunocytochemistry of HUVEC revealed that CN disrupted actin cytoskeleton and altered VE-cadherin distribution at cell–cell contacts. CN downregulated focal adhesion kinase (FAK) and paxillin tyrosine phosphorylation in adherent HUVEC. There was also significant inhibition of angiogenesis in vivo by CN as assessed by implanting Matrigel plugs in C57 mice and measuring ingrowth of blood vessels using either factor VIII staining or hemoglobin determination. In conclusion, the present findings confirm our earlier studies and demonstrate conclusively that CN possess strong anti-angiogenic activity in vitro and in vivo .

  • contortrostatin a dimeric disintegrin from Agkistrodon Contortrix Contortrix inhibits breast cancer progression
    Breast Cancer Research and Treatment, 2000
    Co-Authors: Qing Zhou, Russel P Sherwin, Catherine Parrish, Valda Richters, Susan Groshen, Denice D Tsaowei, Francis S Markland
    Abstract:

    We report the results of a multidisciplinary study on the inhibitory effect of a snake venom disintegrin, contortrostatin, a 13.5 kDa homodimeric protein isolated from Agkistrodon Contortrix Contortrix (southern copperhead) venom, on breast cancer progression. We demonstrate that contortrostatin binds to integrins and blocks the adhesion of human breast cancer cells (MDA-MB-435) to extracellular matrix (ECM) proteins including fibronectin and vitronectin, but it has no effect on adhesion of the cells to laminin and Matrigel. Contortrostatin also prevents invasion of MDA-MB-435 cells through an artificial Matrigel basement membrane. Daily local injection of contortrostatin (5 μg per mouse per day) into MDA-MB-435 tumor masses in an orthotopic xenograft nude mouse model inhibits growth of the tumor by 74% (p = 0.0164). More importantly, it reduces the number of pulmonary macro-metastasis of the breast cancer by 68% (p < 0.001), and micro-metastasis by 62.4% (p < 0.001). Contortrostatin is not cytotoxic to cancer cells, and does not inhibit proliferation of the breast cancer cells in vitro. However, contortrostatin inhibits angiogenesis induced by the breast cancer, as shown by immunohistochemical quantitation of the vascular endothelial cells in tumor tissue removed from the nude mice. We have identified αvβ3, an important integrin mediating cell motility and tumor invasion, as one of the binding sites of contortrostatin on MDA-MB-435 cells. We conclude that contortrostatin blocks αvβ3, and perhaps other integrins, and thus inhibits in vivo progression.

  • contortrostatin a dimeric disintegrin from Agkistrodon Contortrix Contortrix inhibits angiogenesis
    Angiogenesis, 1999
    Co-Authors: Qing Zhou, Marian T Nakada, Catherine Arnold, Kate Y Shieh, Francis S Markland
    Abstract:

    Contortrostatin, a 13.5 kDa disulfide-linked homodimeric polypeptide possessing an Arg–Gly–Asp sequence, was isolated from venom of the southern copperhead snake. Daily injection of contortrostatin into the primary tumor of human breast cancer MDA-MB-435 carried in nude mice significantly inhibited tumor growth and neovascularization of the tumor tissue. On the chick embryo chorioallantoic membrane, contortrostatin inhibited angiogenesis induced by MDA-MB-435 cells, basic fibroblast growth factor, and vascular endothelial growth factor. In addition, contortrostatin effectively blocked adhesion of human umbilical vein endothelial cells (HUVEC) to immobilized vitronectin and significantly inhibited invasion of HUVEC through a Matrigel barrier. Competitive binding assays and adhesion assays with different integrin antibodies suggested that integrin αvβ3 is a binding site for contortrostatin on vascular endothelial cells. Detachment of HUVEC from vitronectin by contortrostatin induced apoptosis. HUVEC adhered and spread well on immobilized contortrostatin without undergoing apoptosis, suggesting that it is the inhibition of adhesion and spreading of HUVEC on extracellular matrix proteins, rather than binding of contortrostatin to integrins per se, that triggers apoptosis. We conclude that contortrostatin binds to αvβ3, and interferes with the anchorage-dependent survival mechanism of the vascular endothelial cells, and the mobility of the cells. The consequent suppression of angiogenesis is an important component of the antineoplastic activity of contortrostatin.

  • resolution of isoforms of natural and recombinant fibrolase the fibrinolytic enzyme from Agkistrodon Contortrix Contortrix snake venom and comparison of their edta sensitivities
    Journal of Chromatography B: Biomedical Sciences and Applications, 1994
    Co-Authors: S L Loayza, Francis S Markland, Mohit Trikha, P Riquelme, J Kuo
    Abstract:

    Abstract Fibrolase, the fibrinolytic enzyme from Agkistrodon Contortrix Contortrix snake venom, is a zinc metalloproteinase with a molecular mass of 23 kDa. We report a method to isolate two isoforms of natural fibrolase (fib1 and fib2) and three isoforms of recombinant fibrolase (r-fib1, r-fib2 and r-fib3) using CM 300 cation-exchange high-performance liquid chromatography. Utilizing mass spectrometry we characterized differences in molecular masses of the isoforms of r-fibrolase. These findings suggest that the isoforms differ by minor sequence variations at their amino-termini. Since the stability of fibrolase is exquisitively sensitive to the removal of zinc, we examined the EDTA sensitivity of the isoforms of fibrolase and r-fibrolase to determine if their different chromatographic behavior is related to differences in their zinc affinities. All of the isoforms examined appear to have similar zinc binding affinities. Thus, the IC50 (concentration of EDTA to produce 50% inhibition of enzymatic activity) for fib1 is 160 μM. For the closely related r-fib1, the IC50 is 180 μM. Similarly, r-fib3 has an IC50 of 140 μM.

Heloisa S Selistredearaujo - One of the best experts on this subject based on the ideXlab platform.

  • effects of acl myotoxin a lys49 phospholipase a2 from Agkistrodon Contortrix laticinctus snake venom on water transport in the isolated toad urinary bladder
    Toxicon, 2004
    Co-Authors: R S Leite, Wilson De Albuquerque Cavalcanti Franco, C L Ownby, Heloisa S Selistredearaujo
    Abstract:

    Abstract 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 μM) 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.

  • expression of an active recombinant lysine 49 phospholipase a2 myotoxin as a fusion protein in bacteria
    Toxicon, 2001
    Co-Authors: C D Giuliani, Dulce Helena Ferreira De Souza, Monica Rosas Da Costa Iemma, A C V Bondioli, L L Ferreira, A C Amaral, Tania F Salvini, Heloisa S Selistredearaujo
    Abstract:

    Abstract 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.

  • isolation and structural characterization of a cytotoxic l amino acid oxidase from Agkistrodon Contortrix laticinctus snake venom preliminary crystallographic data
    Archives of Biochemistry and Biophysics, 1999
    Co-Authors: Dulce Helena Ferreira De Souza, Luiz M Eugenio, Jeffrey E Fletcher, Mingshi Jiang, Richard Charles Garratt, Glaucius Oliva, Heloisa S Selistredearaujo
    Abstract:

    Abstract 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 Mr 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 μg/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 (NH4)2SO4. 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.

Gordon W Schuett - One of the best experts on this subject based on the ideXlab platform.

  • molecular genetic evidence for alternative reproductive strategies in north american pitvipers serpentes viperidae long term sperm storage and facultative parthenogenesis
    Biological Journal of The Linnean Society, 2011
    Co-Authors: Warren Booth, Gordon W Schuett
    Abstract:

    The first documentation of facultative parthenogenesis (FP) in non-avian reptiles (snakes) occurred in 1997, following the application of molecular genetic methods for parentage analysis, and since has been described in only four additional snake species. Here, in two species of live-bearing (viviparous) North American pitviper snakes, using microsatellite DNA fingerprinting, we describe the first record of a virgin birth by FP in the copperhead (Agkistrodon Contortrix), and the first genetically confirmed case of long-term sperm storage (LTSS) of exceptional duration (5 years) in the eastern diamond-backed rattlesnake (Crotalus adamanteus). The capacity of female squamate reptiles (snakes, lizards, and amphisbaenians) to store viable sperm for extended periods of time is a competing hypothesis to FP, and it is often inferred when interpreting certain births where adult females have been isolated from adult male mates for prolonged periods. On reviewing the literature of certain cases of LTSS in snakes, we provide compelling support for the prevalence of FP, casting doubt over the widespread acceptance of LTSS. Accordingly, with the discovery of FP in squamates and the results of the present study, we advocate that in order to differentiate between LTSS and FP, especially under natural conditions, rigorous molecular testing will be required. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104, 934–942.

  • the spatial and reproductive ecology of the copperhead Agkistrodon Contortrix at the northeastern extreme of its range
    Herpetological Monographs, 2009
    Co-Authors: Gordon W Schuett, Charles F Smith, Ryan L Earley, Kurt Schwenk
    Abstract:

    Abstract We studied the spatial and reproductive ecology of a population of Copperheads (Agkistrodon Contortrix), a North American pitviper, in a basalt trap-rock ecosystem in the central Connecticut River Valley, a region that constitutes the northeastern extreme of this species' geographic range. Adult males (n  =  20) and females (n  =  15) were surgically implanted with radio-transmitters and tracked every 48 h during the active season (April through October) for three consecutive years (2001 to 2003). From late autumn to early spring (November through March), when snakes were hibernating and thus inactive, tracking was reduced to once per week. We generated data on movement and other spatial parameters for each subject using GPS coordinates. There were significant sex differences in activity range size and multiple movement parameters. Throughout the active season males had greater activity range sizes and showed greater movement than females. This trend was pronounced during the mating season, which...

  • post fight levels of plasma lactate and corticosterone in male copperheads Agkistrodon Contortrix serpentes viperidae differences between winners and losers
    Physiology & Behavior, 2000
    Co-Authors: Gordon W Schuett, Matthew S Grober
    Abstract:

    During the mating seasons (late summer and spring), male copperheads (Agkistrodon Contortrix; Serpentes, Viperidae) engage in aggressive physical interactions for priority of access to females. These fights generally involve two individuals and are characterized by prominent vertical displays, a high degree of physical contact, and the absence of biting. Ritualized aggression does not occur in females. Although intrasexual aggression in conspecifics has obvious energetic costs (e.g., lactate accumulation) that can affect subsequent behavior, few studies have addressed these costs in reptiles, and no studies have examined snakes. Moreover, recent studies suggest psychoneuroendocrine (catecholamines, glucocorticoids) regulation of metabolism during and following aggressive episodes. There were three main questions addressed in this study. Do winners and losers of staged, pair-wise encounters show differences in post-fight (60-min) levels of plasma lactate and corticosterone (CORT)? Are levels of plasma lactate correlated with levels of plasma CORT? Is fight duration correlated with levels of plasma lactate and CORT? Two different control groups (cage and arena) were used. Body length, body mass, duration of fighting, and season of testing were not correlated with levels of plasma lactate and CORT. At 60-min post-fight, losers had significantly higher levels of mean plasma lactate and CORT when compared to levels in winners and controls, and there were no significant differences between winners and controls. From our results, we suggest the following conclusions. First, elevated levels of CORT in losers, but not winners, result from psychoneuroendocrine factors rather than simple exercise. Second, elevated levels of CORT in losers retard metabolic recovery resulting in higher lactate levels in losers, whereas winners return to pre-fight levels within 60-min post-fight. Last, the CORT response has a net negative effect on metabolic recovery and may be implicated in the protracted suppression of aggressive behavior in losers.

  • annual cycle of plasma testosterone in male copperheads Agkistrodon Contortrix serpentes viperidae relationship to timing of spermatogenesis mating and agonistic behavior
    General and Comparative Endocrinology, 1997
    Co-Authors: Gordon W Schuett, Henry J Harlow, James D Rose, Edward A Van Kirk, William J Murdoch
    Abstract:

    Abstract Reproductive cycles of the majority of squamate reptiles remain undescribed. Few studies are available on seasonal patterns of circulating steroid hormones in snakes. The goal of this study was to document the annual cycle of plasma testosterone (T) in male copperheads, Agkistrodon Contortrix, a North American pitviper (Serpentes, Viperidae). Two experimental conditions were used in this laboratory study. One condition (repeat-test group) consisted of 10 adult males that were sampled once each month for 11 months. The other condition (single-test groups) consisted of 10 groups each with 5 males ( N  = 50), and each male was tested a single time. The single-test condition was used to evaluate whether or not repeated handling and sampling affected T levels. The study was conducted from February–December, 1992. A well-defined seasonal pattern of plasma T levels was detected; patterns were similar under both experimental conditions with the exception that the repeat-test group had slightly lower levels. Levels of T were lowest (baseline) in April–May, increased in early summer (June), and were highest in late summer (August). Thereafter, T levels declined up to the time of hibernation (early November) and changed little during hibernation (November–January). Upon emergence from hibernation in late winter (February), T levels increased sharply from February to March and then decreased from March to April. The results are discussed in the context of timing of spermatogenesis, mating, and male agonistic behavior.

  • fighting dynamics of male copperheads Agkistrodon Contortrix serpentes viperidae stress induced inhibition of sexual behavior in losers
    Zoo Biology, 1996
    Co-Authors: Gordon W Schuett
    Abstract:

    Adult male copperheads (Agkistrodon Contortrix) fight for priority of access to females during the mating periods in spring and late summer. During fights, one male abruptly quits and retreats, and the other chases in pursuit. One male thus emerges as the winner and the other as the loser. Reversal of this outcome does not occur during the time of observation (30 min) nor in 24 hr postfight trials. In all cases, winners gain priority of access to females. Losers, in contrast, do not pursue females nor gain access to them, even when winners are removed from the arena. In this study, courtship performance of male A. Contortrix was studied in the laboratory using subjects with either winning or losing experience from staged fights. All males used first were tested with a single female to determine courtship performance prior to the agonistic trials. From these tests, each male was given a single courtship score of 0 (no courtship) to 3 (most intense courtship). Only males receiving a score of 2–3 were used in the initial agonistic trials. All staged fights were conducted in a large arena and involved two males and one female. Following fights, winners and losers were tested again for courtship performance. In trials conducted at 24 hr and 7 days postfight, only losers were tested. It was found that prefight courtship scores were not significantly different between winners and losers. At 30 min postfight, most losers showed complete suppression of courtship behavior (score 0). Winners, in contrast, showed equivalent or an increase in their courtship scores. At 24 hr postfight, courtship scores of losers remained significantly lower than their prefight scores. At 7 days postfight, courtship scores of losers were not significantly difference from their original prefight scores. Fighting behavior in free-ranging A. Contortrix represents a potentially significant cost to losers if it is associated with loss of reproductive opportunities. Recent evidence from hormonal studies indicates that inhibition of courtship and fighting behavior in male A. Contortrix is stress-induced. © 1996 Wiley-Liss, Inc.

Ribeiro, Juliana Uema - One of the best experts on this subject based on the ideXlab platform.

  • Obtenção de uma desintegrina recombinante de Agkistrodon Contortrix laticinctus e estudo dos efeitos de desintegrinas na expressão do fator de crescimento de endotélio vascular.
    Programa de Pós-Graduação em Genética Evolutiva e Biologia Molecular - PPGGEv, 2005
    Co-Authors: Ribeiro, Juliana Uema
    Abstract:

    Disintegrins are snake venom proteins used in the study of integrin-mediated cellular adhesion through integrins. Recombinant disintegrin production may notable supply faster, larger and most homogeneous protein samples than bruit venom processing. This work describes optimization of the recombinant ACLD-C expression technique. ACLD-C is a disintegrin-like protein from Agkistrodon Contortrix laticinctus, that have a disintegrin-like domain with DCD motif, and a cysteine-rich domain. This protein was subcloned from the ACLD clone (GenBank U86634), a metallopeptidase. A protocol of expression was established in our laboratory, where recombinant ACLD-C is produced in E. coli, using the expression vector pMal-p. This allows protein target expression in form of N-terminal fusion with MBP (maltose-binding protein), that facilitate purification, correct folding and production of disintegrin in soluble form. In fact, MBP/ACLD-C was was expressed in soluble form and purified in two stages: affinity comatography in amilose resin and cromatography of gel-filtration in superdex-200 resin. Modifications in expression protocol optimized MBP/ACLD-C expression in culture of 0,4 mg/L of culture of 1,2 mg/L (12,6% total of protein in periplasmic space of E.coli). MBP/ACLD-C imobilizated in amilose resin was subjected to digestion with enzyme factor Xa to separate ACLD-C of MBP. The separation was confirmed through positive reaction with anti-ALT-C antibody. ALT-C is a disintegrin-like protein from Bothrops alternatus. ALT-C induced both expression of VEGF in human fibroblasts and proliferation of endothelial cells and angiogenesis. In second part of this work, we investigated if MBP/ACLD-C was also able to induce the expression of this growth factor in this cells. MBP/ACLD-C and two other disintegrins, echistatina, a disintegrin-RGD, and EC6, a heterodimeric disintegrin, were incubated with fibroblasts. The influency of these proteins in VEGF expression was evaluated by ELISA. MBP/ACLD-C was able to strongly induce the expression of VEGF until 4 hours, when the level decreased until 24 hours. Experiment in period of 24 48 hours was not done due to problems of contaminations. Echistatin did not induced VEGF expression, but induced fibroblasts dettachment from plastic. The latter event was not observed in MBP/ACLD-C or EC6 incubated cells.Universidade Federal de Minas GeraisAs desintegrinas são proteínas de veneno de serpente usadas no estudo de processos biológicos envolvendo a adesão celular mediada por integrinas. A produção de desintegrinas recombinantes é de grande importância para tais estudos, pois podem ser obtidas de forma mais rápida, com maior rendimento e qualidade mais constante quando comparada com a obtenção a partir de veneno bruto. Esta dissertação descreve a otimização da expressão da ACLD-C recombinante. ACLD-C é uma proteína tipo desintegrina da serpente Agkistrodon Contortrix laticinctus, que possui um domínio desintegrina-like com um motivo DCD e um domínio rico em cisteína. Esta proteína foi subclonada a partir do clone da ACLD (GenBank U86634), uma metalopeptidase. Um protocolo de expressão foi estabelecido em nosso laboratório, onde ACLD-C recombinante é produzida em E. coli, utilizando-se o vetor de expressão pMal-p. Este vetor possibilita a expressão da proteína-alvo na forma de fusão Nterminal com a proteína MBP (maltose-binding protein), a qual facilita a purificação, o enovelamento correto e a produção da desintegrina na forma solúvel. De fato, MBP/ACLD-C foi expressa na forma solúvel e purificada em duas etapas: cromatografia de afinidade em resina de amilose e cromatografia de gel-filtração em resina superdex-200. Modificações no protocolo de expressão permitiram a otimização da expressão da MBP/ACLD-C de 0,4mg/L de cultura para 1,2mg/L de cultura O rendimento final da MBP/ACLD-C foi de 1,2mg/L de cultura (12,6% do total de proteínas no espaço periplásmico de E. coli). MBP/ACLD-C imobilizada em resina de amilose foi submetida à reação de clivagem com a enzima fator Xa para separar ACLD-C da MBP. A separação foi confirmada através da reação positiva ao anticorpo anti-ALT-C, uma desintegrina-like de Bothrops alternatus. Estudos com ALT-C mostraram que ela foi capaz de induzir a expressão de VEGF (vascular endothelial growth factor) em fibroblastos humanos e induzir a proliferação de células endoteliais e a angiogênese. Assim, resolveu-se investigar se MBP/ACLD-C também é capaz de induzir a expressão deste fator de crescimento nestas células, o que constitui a segunda parte deste trabalho. MBP/ACLD-C e outras duas desintegrinas, echistatina, uma desintegrina-RGD, e EC6, uma desintegrina heterodimérica, foram incubadas com os fibroblastos para verificar a influência dessas proteínas na expressão de VEGF através de ensaios de ELISA. MBP/ACLD-C foi capaz de induzir fortemente a expressão de VEGF até 4h, quando o nível da citocina decresceu até 24 e novamente aumentou até 48h. EC6 também foi capaz de induzir a expressão do fator de crescimento até 4h, quando o nível também decresceu até 24h. O experimento no período de 24h-48h não foi realizado devido a problemas de contaminação. Echistatina, por sua vez, não apresentou indução de VEGF em relação ao controle, e as células incubadas com essa desintegrina desaderiram do plástico, o que não ocorreu com as outras duas proteínas

Caroline Krieger De Moraes - One of the best experts on this subject based on the ideXlab platform.

  • Efeito da rACLF, uma metalopeptidase recombinante da peçonha de Agkistrodon Contortrix laticinctus, na viabilidade celular, expressão de citocinas e degradação de proteínas da matriz extracelular.
    Universidade Federal de São Carlos, 2006
    Co-Authors: Caroline Krieger De Moraes
    Abstract:

    A ACLF é uma metalopeptidase da peçonha da serpente Agkistrondon Contortrix laticinctus. A ACLF é sintetizada na forma de zimogênio (pró-ACLF) com 412 aminoácidos, enquanto a sua forma ativa possui 222 aminoácidos. A fase aberta de leitura que codifica para a peptidase foi isolada (de uma biblioteca de cDNA feita a partir da glândula venenífera da serpente) e caracterizada. Este trabalho apresenta os efeitos da rACLF em células endoteliais (HUVECs), tumorais (HeLa, MDA-MB-231 e MCF-7) e não-tumorais (fibroblastos), bem como a atividade proteolítica da enzima sobre proteínas da matriz extracelular, como colágeno, fibronectina, laminina e trombospondina. Nossos resultados demonstraram que a rACLF inibiu a apoptose induzida por privação de soro fetal bovino em HUVECs, mas não foi capaz de induzir a proliferação celular. A rACLF apresentou efeito direto sobre as células endoteliais, aumentando a liberação de IL-8, além de induzir a expressão de moléculas envolvidas na adesão celular e reposta inflamatória. A rACLF e a rACLH não foram citotóxicas para fibroblastos humanos. Por outro lado as duas enzimas estudadas causaram redução na viabilidade alteração na morfologia e desgrudamento em células HeLa. Em fibroblastos, a rACLF modulou a expressão das quimoquinas CXC, IL-8 e GRO e CC, MCP1. Nos sobrenadantes de fibroblastos e HeLa incubados com rACLF por 48 h não foi observada diferença na secreção de MMP-2 (gelatinase-A). A rACLF apresentou atividade sobre proteínas da matriz extracelular, como laminina, fibronectina, colágeno IV e trombospondina. A rACLF também hidrolisou alguns substratos fluorogênicos e foi mais eficiente na hidrólise do substrato correspondente ao peptídeo central da cadeia β da insulina, quando comparada com a rACLH .Os resultados deste trabalho contribuíram para melhor entendimento dos efeitos da proteína estudada sobre diferentes linhagens celulares e abrem perspectivas para novos estudos voltados à elucidação dos mecanismos de regulação e sinalização afetados pela atividade da proteína.ACLF is a fibrinolytic non-hemorrhagic metallopeptidase from the venom of the snake Agkistrodon Contortrix laticinctus. rACLF is synthesized as a zymogen (pro- ACLF) with 412 aminoacids, while the active form of enzyme has 222 aminoacids. The ORF (open reading frame) that codes for the peptidase was isolated (from a cDNA venom gland library of A. Contortrix. lacticinctus ) and characterized. This work shows the effects of rACLF on endothelial cells (HUVECs), tumor cells (HeLa, MDA-MB-231 and MCF-7) and non-tumor cell (fibroblast) as well its proteolytic activity on extracellular matrix proteins. Our results showed that rACLF inhibited the apoptosis induced by serum deprivation in HUVECs, but had no effect on cell proliferation. rACLF had direct effect on HUVECs, since it was observed an increased amount of IL-8 in the cell supernatants, in addition to the expression induction of molecules related to adhesion and inflammation processes. rACLF and rACLH were not cytotoxic to human fibroblasts. On the other hand, both proteins caused decrease of cell viability, changes in morphology, and detachment of HeLa cells. On human fibroblasts, rACLF modulated the expression of chemokines CXC, IL-8 and GRO, and chemokine CC, MCP1. The supernatant of human fibroblasts and HeLa cells were analyzed for MMP-2 (gelatinase-A) secretion. No difference was observed after 48 h incubation with rACLF and controls. rACLF presented hydrolytic activity on extracellular matrix proteins, such as laminin, fibronectin, collagen IV, and thrombospondin. Furthermore, hydrolytic activity on insulin β chain peptide was tested and compared to rACLH activity. rACLF was shown to be more efficient than rACLH on this substrate. In conclusion, the results of this study increased the knowledge on the protein effects on different cell lines and open up perspectives for new studies concerning signaling and regulation mechanisms affected by this protein activity

  • Efeito da rACLF, uma metalopeptidase recombinante da peçonha de Agkistrodon Contortrix laticinctus, na viabilidade celular, expressão de citocinas e degradação de proteínas da matriz extracelular.
    Programa de Pós-graduação em Ciências Fisiológicas, 2006
    Co-Authors: Caroline Krieger De Moraes
    Abstract:

    ACLF is a fibrinolytic non-hemorrhagic metallopeptidase from the venom of the snake Agkistrodon Contortrix laticinctus. rACLF is synthesized as a zymogen (pro- ACLF) with 412 aminoacids, while the active form of enzyme has 222 aminoacids. The ORF (open reading frame) that codes for the peptidase was isolated (from a cDNA venom gland library of A. Contortrix. lacticinctus ) and characterized. This work shows the effects of rACLF on endothelial cells (HUVECs), tumor cells (HeLa, MDA-MB-231 and MCF-7) and non-tumor cell (fibroblast) as well its proteolytic activity on extracellular matrix proteins. Our results showed that rACLF inhibited the apoptosis induced by serum deprivation in HUVECs, but had no effect on cell proliferation. rACLF had direct effect on HUVECs, since it was observed an increased amount of IL-8 in the cell supernatants, in addition to the expression induction of molecules related to adhesion and inflammation processes. rACLF and rACLH were not cytotoxic to human fibroblasts. On the other hand, both proteins caused decrease of cell viability, changes in morphology, and detachment of HeLa cells. On human fibroblasts, rACLF modulated the expression of chemokines CXC, IL-8 and GRO, and chemokine CC, MCP1. The supernatant of human fibroblasts and HeLa cells were analyzed for MMP-2 (gelatinase-A) secretion. No difference was observed after 48 h incubation with rACLF and controls. rACLF presented hydrolytic activity on extracellular matrix proteins, such as laminin, fibronectin, collagen IV, and thrombospondin. Furthermore, hydrolytic activity on insulin β chain peptide was tested and compared to rACLH activity. rACLF was shown to be more efficient than rACLH on this substrate. In conclusion, the results of this study increased the knowledge on the protein effects on different cell lines and open up perspectives for new studies concerning signaling and regulation mechanisms affected by this protein activity.Universidade Federal de Minas GeraisA ACLF é uma metalopeptidase da peçonha da serpente Agkistrondon Contortrix laticinctus. A ACLF é sintetizada na forma de zimogênio (pró-ACLF) com 412 aminoácidos, enquanto a sua forma ativa possui 222 aminoácidos. A fase aberta de leitura que codifica para a peptidase foi isolada (de uma biblioteca de cDNA feita a partir da glândula venenífera da serpente) e caracterizada. Este trabalho apresenta os efeitos da rACLF em células endoteliais (HUVECs), tumorais (HeLa, MDA-MB-231 e MCF-7) e não-tumorais (fibroblastos), bem como a atividade proteolítica da enzima sobre proteínas da matriz extracelular, como colágeno, fibronectina, laminina e trombospondina. Nossos resultados demonstraram que a rACLF inibiu a apoptose induzida por privação de soro fetal bovino em HUVECs, mas não foi capaz de induzir a proliferação celular. A rACLF apresentou efeito direto sobre as células endoteliais, aumentando a liberação de IL-8, além de induzir a expressão de moléculas envolvidas na adesão celular e reposta inflamatória. A rACLF e a rACLH não foram citotóxicas para fibroblastos humanos. Por outro lado as duas enzimas estudadas causaram redução na viabilidade alteração na morfologia e desgrudamento em células HeLa. Em fibroblastos, a rACLF modulou a expressão das quimoquinas CXC, IL-8 e GRO e CC, MCP1. Nos sobrenadantes de fibroblastos e HeLa incubados com rACLF por 48 h não foi observada diferença na secreção de MMP-2 (gelatinase-A). A rACLF apresentou atividade sobre proteínas da matriz extracelular, como laminina, fibronectina, colágeno IV e trombospondina. A rACLF também hidrolisou alguns substratos fluorogênicos e foi mais eficiente na hidrólise do substrato correspondente ao peptídeo central da cadeia β da insulina, quando comparada com a rACLH .Os resultados deste trabalho contribuíram para melhor entendimento dos efeitos da proteína estudada sobre diferentes linhagens celulares e abrem perspectivas para novos estudos voltados à elucidação dos mecanismos de regulação e sinalização afetados pela atividade da proteína