RESUMEN
In yeast, as in other eukaryotes, calcium plays an essential role in signaling transduction to regulate different processes. Many pieces of evidence suggest that glucose-induced activation of plasma membrane H+-ATPase, essential for yeast physiology, is related to calcium signaling. Until now, no protein that could be regulated by calcium in this context has been identified. Lpx1p, a serine-protease that is also involved in the glucose-induced activation of the plasma membrane H+-ATPase, could be a candidate to respond to intracellular calcium signaling involved in this process. In this work, by using different approaches, we obtained many pieces of evidence suggesting that the requirement of calcium signaling for activation of the plasma membrane H+-ATPase is due to its requirement for activation of Lpx1p. According to the current model, activation of Lpx1p would cause hydrolysis of an acetylated tubulin that maintains the plasma membrane H+-ATPase in an inactive state. Therefore, after its activation, Lpx1p would hydrolyze the acetylated tubulin making the plasma membrane H+-ATPase accessible for phosphorylation by at least one protein kinase.
Asunto(s)
Señalización del Calcio , Membrana Celular/metabolismo , Glucosa/metabolismo , Fosfolipasas A/metabolismo , ATPasas de Translocación de Protón/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Calcio/metabolismo , Citosol/metabolismo , Regulación Fúngica de la Expresión Génica , ProteolisisRESUMEN
The myotoxic mechanism for PLA2-like toxins has been proposed recently to be initiated by an allosteric change induced by a fatty acid binding to the protein, leading to the alignment of the membrane docking site (MDoS) and membrane disrupting site (MDiS). Previous structural studies performed by us demonstrated that MjTX-II, a PLA2-like toxin isolated from Bothrops moojeni, presents a different mode of ligand-interaction caused by natural amino acid substitutions and an insertion. Herein, we present four crystal structures of MjTX-II, in its apo state and complexed with fatty acids of different lengths. Analyses of these structures revealed slightly different oligomeric conformations but with both MDoSs in an arrangement that resembles an active-state PLA2-like structure. To explore the structural transitions between apo protein and fatty-acid complexes, we performed Normal Mode Molecular Dynamics simulations, revealing that oligomeric conformations of MjTX-II/fatty acid complexes may be reached in solution by the apo structure. Similar simulations with typical PLA2-like structures demonstrated that this transition is not possible without the presence of fatty acids. Thus, we hypothesize that MjTX-II does not require fatty acids to be active, although these ligands may eventually help in its stabilization by the formation of hydrogen bonds. Therefore, these results complement previous findings for MjTX-II and help us understand its particular ligand-binding properties and, more importantly, its particular mechanism of action, with a possible impact on the design of structure-based inhibitors for PLA2-like toxins in general.
Asunto(s)
Ácidos Grasos/química , Simulación de Dinámica Molecular , Fosfolipasas A/química , Conformación Proteica , Multimerización de Proteína , Animales , Bothrops/metabolismo , Biología Computacional/métodos , Cristalografía por Rayos X , Ácidos Grasos/metabolismo , Enlace de Hidrógeno , Ligandos , Fosfolipasas A/metabolismo , Unión ProteicaRESUMEN
Local myonecrosis resulting from snakebite envenomation is not efficiently neutralized by regular antivenom administration. This limitation is considered to be a significant health problem by the World Health Organization. Phospholipase A2-like (PLA2-like) proteins are among the most important proteins related to the muscle damage resulting from several snake venoms. However, despite their conserved tertiary structure compared with PLA2s, their biological mechanism remains incompletely understood. Different oligomeric conformations and binding sites have been identified or proposed, leading to contradictory data in the literature. In the last few years, a comprehensive hypothesis has been proposed based on fatty-acid binding, allosteric changes and the presence of two different interaction sites. In the present study, a combination of techniques were used to fully understand the structural-functional characteristics of the interaction between suramin and MjTX-II (a PLA2-like toxin). In vitro neuromuscular studies were performed to characterize the biological effects of the protein-ligand interaction and demonstrated that suramin neutralizes the myotoxic activity of MjTX-II. The high-resolution structure of the complex identified the toxin-ligand interaction sites. Calorimetric assays showed two different binding events between the protein and the inhibitor. It is demonstrated for the first time that the inhibitor binds to the surface of the toxin, obstructing the sites involved in membrane docking and disruption according to the proposed myotoxic mechanism. Furthermore, higher-order oligomeric formation by interaction with interfacial suramins was observed, which may also aid the inhibitory process. These results further substantiate the current myotoxic mechanism and shed light on the search for efficient inhibitors of the local myonecrosis phenomenon.
Asunto(s)
Antivenenos/farmacología , Bothrops/metabolismo , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/metabolismo , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/metabolismo , Suramina/farmacología , Animales , Sitios de Unión , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Venenos de Crotálidos/química , Venenos de Crotálidos/toxicidad , Cristalografía por Rayos X , Masculino , Ratones , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fosfolipasas A/química , Fosfolipasas A/toxicidadRESUMEN
O objetivo deste artigo é investigar relações entre renda e escolaridade com condições de saúde e nutrição em obesos graves. Estudo transversal ambulatorial com 79 pacientes de primeira consulta, com Índice de Massa Corporal (IMC) ≥ 35 kg/m2 e idade ≥ 20 anos. Coletaram-se dados: sociodemográficos, antropométricos, estilo de vida, exames bioquímicos e consumo alimentar. O IMC médio foi 48,3 ± 6,9 kg/m2. Observou-se correlação negativa significante de escolaridade com variáveis peso (r = -0,234) e IMC (r = -0,364) e de renda familiar per capita com consumo diário de vegetal A (r = -0,263). Após análise multivariada maior renda familiar per capita se associou à ausência de cardiopatia (RP: 0,51, IC95%: 0,32-0,81), maior consumo diário de vegetal A (RP: 1,79, IC95%: 1,16-2,75) e doces (RP: 3,12, IC95%: 1,21-8,04). Em obesos graves a maior renda familiar per capita se associou à ausência de cardiopatia e maior consumo de vegetais folhosos e doces. Já a escolaridade não se manteve associada às condições de saúde e nutrição.
This article seeks to investigate the relationship between income and educational level and health and nutritional conditions among the morbidly obese. A cross-sectional study was conducted with 79 patients at first appointment, with Body Mass Index (BMI) ≥ 35 kg/m2 and age ≥ 20 years. The following data was collected: demographic, socioeconomic, anthropometric, lifestyle, biochemical and food intake data. Average BMI was 48.3 ± 6.9 kg/m2. There was a significant negative correlation between education level and the variables of weight (r = -0.234) and BMI (r = -0.364) and per capita family income with daily consumption of leafy vegetables (r = -0.263). After multivariate analysis, higher per capita family income was associated with the absence of heart disease (PR: 0.51, CI95%: 0.32-0.81), higher daily consumption of leafy vegetables (PR: 1.79, CI95%: 1.16-2.75) and candy (PR: 3.12, CI95%: 1.21-8.04). In the morbidly obese, per capita household income was associated with absence of heart disease and higher consumption of leafy vegetables and candy. On the other hand, education level was not associated with health and nutrition conditions.
Asunto(s)
Arabidopsis/enzimología , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Fosfolipasas A/metabolismo , /farmacología , /farmacología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Glucuronidasa/metabolismo , Luciferasas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfolipasas A/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Plantones/efectos de los fármacos , Plantones/metabolismo , Factores de TiempoRESUMEN
Este estudo objetivou compreender as práticas de cuidado dos profissionais de saúde que assistem os idosos Kaingang. Estudo qualitativo, apoiado na etnografia, realizado com dez profissionais à que atuam na atenção primária saúde da Terra Indígena Faxinal, Paraná, Brasil. Os dados foram coletados no período de novembro de 2010 a fevereiro de 2012 por meio da observação participante e entrevistas, e, analisados à luz da Teoria Transcultural do Cuidado. Identificaram-se como práticas de cuidado a medicação e imunização, bem como, cuidados da medicina tradicional. Para realização destes cuidados, os profissionais dispunham de estratégias que proporcionavam manutenção dos idosos na assistência. Conclui-se que valores culturais e científicos necessitam integrar a assistência para melhoria da saúde dos idosos indígenas.
This research aims to understand the care practices of health professionals who assist the elderly Kaingang. It is a qualitative study, supported in ethnography, conducted by ten professionals working in primary health care in the indigenous land of Faxinal, Paraná, Brazil. The data was collected from November 2010 to February 2012 by participant observation and interviews, and analyzed based on the Transcultural Care Theory. Was identified the preoccupation of the carers practices with the medication and immunization, as well as traditional medical care. To achieve these, care professionals had strategies that implemented maintenance of older people in care. We conclude that cultural values and integrate scientific need assistance to improve the health of elderly indigenous.
Este estudio tuvo como objetivo entender las prácticas de cuidado de los profesionales de la salud que asisten a los ancianos Kaingang. Estudio cualitativo, apoyado en la etnografía, llevado a cabo con diez profesionales que trabajan en la atención primaria de la salud de la tierra indígena de Faxinal, Paraná, Brasil. Los datos fueron recogidos a partir de noviembre 2010 a febrero 2012 a través de la observación participante y las entrevistas, y analizado con base en la Teoría del Cuidado Transcultural. Se identificaron las prácticas de atención médica y imunizacion,el cuidado de la medicina, así tradicional. Para lograrlo, los profesionales tenían estrategias que proporcionaban el mantenimiento de las personas mayores en su atención. Se concluye que los valores culturales y científicos necesitan ayuda para mejorar la salud de los ancianos indígenas.
Asunto(s)
Animales , Ratas , Hígado/enzimología , Lisosomas/enzimología , Fosfolipasas A/metabolismo , Fosfolipasas/metabolismo , Inhibidores de Proteasas/farmacología , Células Cultivadas , Quimotripsina/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , Leucina/análogos & derivados , Leucina/farmacología , Leupeptinas/farmacología , Oligopéptidos/farmacología , Pepstatinas/farmacología , Fosfolipasas A1 , Factores de TiempoRESUMEN
The prominent local myotoxic effects induced by Bothrops snake venom are due, in part, to myotoxins. This effect is not neutralized by antivenom, which is the main therapy for victims of snakebite. Two basic myotoxins named MjTX-I and MjTX-II were isolated from Bothrops moojeni venom. Both myotoxins have a Lys-49 phospholipase A2 structure devoid of enzymatic activity, but are highly myonecrotic and edema-inducing. In this study, we analyzed the effect of a low-level laser (LLL) at 685 nm, an energy density of 2.2 J cm(-2), and the irradiation time of 15 s, and a light emitting diode (LED) at 635 or 945 nm at energy densities of 4 and 3.8 J cm(-2), and irradiation times of 41 and 38 s, respectively, applied 30 min and 3 h after edema formation in mice caused by MjTX-I or MjTX-II. MjTX-I or MjTX-II caused a significant edema formation in envenomed paws. LLL and LED irradiation significantly reduced the edema formation by both myotoxins from 1 up to 6 hours after the injection. Both LLL and LEDs were similar in reducing the edema formation induced by myotoxins. The combined photobiostimulation with antivenom had the same effect in reducing edema as treatment with the LLL or LEDs alone. In conclusion, the results of this study indicate that photobiostimulation could be used in association with antivenom therapy for treatment of local effects of Bothrops species venom.
Asunto(s)
Bothrops/metabolismo , Edema/inducido químicamente , Fosfolipasas A/toxicidad , Ponzoñas/metabolismo , Animales , Edema/radioterapia , Terapia por Luz de Baja Intensidad , Masculino , Ratones , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/metabolismoRESUMEN
The neurotoxic effects produced by a tentacle venom extract and a fraction were analyzed and correlated by in vivo and in vitro approaches. The tentacle venom extract exhibited a wide range of protein components (from 24 to >225 kDa) and produced tetanic reactions, flaccid paralysis, and death when injected into crabs. Two chromatography fractions also produced uncontrolled appendix movements and leg stretching. Further electrophysiological characterization demonstrated that one of these fractions potently inhibited ACh-elicited currents mediated by both vertebrate fetal and adult muscle nicotinic acetylcholine receptors (nAChR) subtypes. Receptor inhibition was concentration-dependent and completely reversible. The calculated IC(50) values were 1.77 µg/µL for fetal and 2.28 µg/µL for adult muscle nAChRs. The bioactive fraction was composed of a major protein component at ~90 kDa and lacked phospholipase A activity. This work represents the first insight into the interaction of jellyfish venom components and muscle nicotinic receptors.
Asunto(s)
Venenos de Cnidarios/toxicidad , Neurotoxinas/toxicidad , Receptores Nicotínicos/fisiología , Escifozoos , Animales , Conducta Animal/efectos de los fármacos , Braquiuros/efectos de los fármacos , Braquiuros/fisiología , Venenos de Cnidarios/química , Masculino , Ratones , Músculos/metabolismo , Neurotoxinas/química , Oocitos/efectos de los fármacos , Oocitos/fisiología , Fosfolipasas A/metabolismo , Xenopus laevisRESUMEN
Iron accumulation and oxidative stress are hallmarks of retinas from patients with age-related macular degeneration (AMD). We have previously demonstrated that iron-overloaded retinas are a good in vitro model for the study of retinal degeneration during iron-induced oxidative stress. In this model we have previously characterized the role of cytosolic phospholipase A2 (cPLA2) and calcium-independent isoform (iPLA2). The aim of the present study was to analyze the implications of Group V secretory PLA2 (sPLA2), another member of PLA2 family, in cyclooxygenase (COX)-2 and nuclear factor kappa B (NF-κB) regulation. We found that sPLA2 is localized in cytosolic fraction in an iron concentration-dependent manner. By immunoprecipitation (IP) assays we also demonstrated an increased association between Group V sPLA2 and COX-2 in retinas exposed to iron overload. However, COX-2 activity in IP assays was observed to decrease in spite of the increased protein levels observed. p65 (RelA) NF-κB levels were increased in nuclear fractions from retinas exposed to iron. In the presence of ATK (cPLA2 inhibitor) and YM 26734 (sPLA2 inhibitor), the nuclear localization of both p65 and p50 NF-κB subunits was restored to control levels in retinas exposed to iron-induced oxidative stress. Membrane repair mechanisms were also analyzed by studying the participation of acyltransferases in phospholipid remodeling during retinal oxidation stress. Acidic phospholipids, such as phosphatidylinositol (PI) and phosphatidylserine (PS), were observed to show an inhibited acylation profile in retinas exposed to iron while phosphatidylethanolamine (PE) showed the opposite. The use of PLA2 inhibitors demonstrated that PS is actively deacylated during iron-induced oxidative stress. Results from the present study suggest that Group V sPLA2 has multiple intracellular targets during iron-induced retinal degeneration and that the specific role of sPLA2 could be related to inflammatory responses by its participation in NF-κB and COX-2 regulation.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Fosfolipasas A2 Grupo V/fisiología , Degeneración Macular/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Retina/efectos de los fármacos , Acetilación , Acetiltransferasas/metabolismo , Animales , Western Blotting , Bovinos , Citosol/metabolismo , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Compuestos Ferrosos/toxicidad , Fosfolipasas A2 Grupo V/antagonistas & inhibidores , Sobrecarga de Hierro/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositoles/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipasas A/metabolismo , Fosfolipasas A/fisiología , Retina/metabolismoRESUMEN
BACKGROUND: Glucose induces H(+)-ATPase activation in Saccharomyces cerevisiae. Our previous study showed that (i) S. cerevisiae plasma membrane H(+)-ATPase forms a complex with acetylated tubulin (AcTub), resulting in inhibition of the enzyme activity; (ii) exogenous glucose addition results in the dissociation of the complex and recovery of the enzyme activity. METHODS: We used classic biochemical and molecular biology tools in order to identify the key components in the mechanism that leads to H(+)-ATPase activation after glucose treatment. RESULTS: We demonstrate that glucose-induced dissociation of the complex is due to pH-dependent activation of a protease that hydrolyzes membrane tubulin. Biochemical analysis identified a serine protease with a kDa of 35-40 and an isoelectric point between 8 and 9. Analysis of several knockout yeast strains led to the detection of Lpx1p as the serine protease responsible of tubulin proteolysis. When lpx1Δ cells were treated with glucose, tubulin was not degraded, the AcTub/H(+)-ATPase complex did not undergo dissociation, and H(+)-ATPase activation was significantly delayed. CONCLUSION: Our findings indicate that the mechanism of H(+)-ATPase activation by glucose involves a decrease in the cytosolic pH and consequent activation of a serine protease that hydrolyzes AcTub, accelerating the process of the AcTub/H(+)-ATPase complex dissociation and the activation of the enzyme. GENERAL SIGNIFICANCE: Our data sheds light into the mechanism by which acetylated tubulin dissociates from the yeast H(+)-ATPase, identifying a degradative step that remained unknown. This finding also proposes an indirect way to pharmacologically regulate yeast H(+)-ATPase activity and open the question about mechanistic similarities with other higher eukaryotes.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Glucosa/farmacología , Proteínas de la Membrana/metabolismo , Fosfolipasas A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Serina Proteasas/metabolismo , Tubulina (Proteína)/metabolismo , Acetilación/efectos de los fármacos , Adenosina Trifosfatasas/genética , Membrana Celular/enzimología , Membrana Celular/genética , Activación Enzimática/efectos de los fármacos , Proteínas de la Membrana/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Fosfolipasas A/genética , Proteínas de Saccharomyces cerevisiae/genética , Serina Proteasas/genética , Tubulina (Proteína)/genéticaRESUMEN
The aim of the present research was to analyze the pathways for phosphatidic acid metabolism in purified nuclei from liver. Lipid phosphate phosphatase, diacylglycerol lipase, monoacylglycerol lipase and PA-phospholipase type A activities were detected. The presence of lysophosphatidic acid significantly reduced DAG production while sphingosine 1-phoshate and ceramide 1-phosphate reduced MAG formation from PA. Using different enzymatic modulators (detergents and ions) an increase in the PA metabolism by phospholipase type A was observed. Our findings evidence an active PA metabolism in purified liver nuclei which generates important lipid second messengers, and which could thus be involved in nuclear processes such as gene transcription.
Asunto(s)
Núcleo Celular/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Ácidos Fosfatidicos/metabolismo , Animales , Calcio/farmacología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Ceramidas/metabolismo , Diglicéridos/metabolismo , Immunoblotting , Lipoproteína Lipasa/metabolismo , Lisofosfolípidos/metabolismo , Magnesio/farmacología , Masculino , Microscopía Electrónica , Monoacilglicerol Lipasas/metabolismo , Monoglicéridos/metabolismo , Octoxinol/farmacología , Fosfatidato Fosfatasa/metabolismo , Fosfolipasas A/metabolismo , Ratas , Ratas Wistar , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
Venom (10-100 µg/ml) from Bothrops alcatraz, a pitviper from the Alcatrazes Archipelago off the coast of southeastern Brazil, caused progressive, irreversible neuromuscular blockade in chick isolated biventer cervicis preparations. The venom also inhibited contractures to exogenous ACh (110 µM) and KCl (20 mM), caused myofiber damage and increased creatine kinase release. Commercial bothropic antivenom raised against mainland Bothrops species neutralized the neuromuscular activity, depending on the venom concentration.
Asunto(s)
Bothrops , Bloqueo Neuromuscular , Venenos de Serpiente/toxicidad , Acetilcolina/efectos adversos , Animales , Antivenenos/farmacología , Brasil , Pollos , Creatina Quinasa/metabolismo , Masculino , Músculo Esquelético/efectos de los fármacos , Miofibrillas/efectos de los fármacos , Miofibrillas/patología , Fosfolipasas A/metabolismo , Nervio Frénico , Cloruro de Potasio/efectos adversos , Receptores Nicotínicos/metabolismoRESUMEN
The red alga Gracilaria chilensis is commercially farmed for the production of agar hydrocolloids, but some susceptible algae in farms suffer from intense epiphyte growth. We investigated the induced chemical defense response of G. chilensis against epiphytes and demonstrated that an extract of an epiphyte-challenged alga can trigger a defense response. The hormonally active metabolites were purified by RP-HPLC. Treatment with the extract or the purified fraction changed the chemical profile of the alga and increased resistance against epiphyte spores. Semi-quantitative RT-PCR and enzyme assays demonstrated that this metabolic response occurs after an increase in lipoxygenase and phospholipase A2 activity. Although this suggests the involvement of regulatory oxylipins, neither jasmonic acid nor the algal metabolite prostaglandin E2 triggers comparable defense responses.
Asunto(s)
Gracilaria/enzimología , Lipooxigenasa/metabolismo , Fosfolipasas A/metabolismo , Inmunidad de la Planta/fisiología , Ciclopentanos/metabolismo , Dinoprostona/metabolismo , Lipooxigenasa/genética , Oxilipinas/metabolismo , Fosfolipasas A/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/fisiologíaRESUMEN
Phospholipases A(2) (PLA(2)s) are one of the main components of bothropic venoms; in addition to their phospholipid hydrolysis action, they are involved in a wide spectrum of pharmacological activities, including neurotoxicity, myotoxicity and cardiotoxicity. Caffeic acid is an inhibitor that is present in several plants and is employed for the treatment of ophidian envenomations in the folk medicine of many developing countries; as bothropic snake bites are not efficiently neutralized by conventional serum therapy, it may be useful as an antivenom. In this work, the cocrystallization and preliminary X-ray diffraction analysis of the Lys49-PLA(2) piratoxin I from Bothrops pirajai venom in the presence of the inhibitor caffeic acid (CA) are reported. The crystals diffracted X-rays to 1.65â Å resolution and the structure was solved by molecular-replacement techniques. The electron-density map unambiguously indicated the presence of three CA molecules that interact with the C-terminus of the protein. This is the first time a ligand has been observed bound to this region and is in agreement with various experiments previously reported in the literature.
Asunto(s)
Bothrops/metabolismo , Ácidos Cafeicos/metabolismo , Venenos de Crotálidos/química , Fosfolipasas A2 Grupo II/química , Animales , Cristalización , Cristalografía por Rayos X/métodos , Ligandos , Modelos Moleculares , Fosfolipasas A/química , Fosfolipasas A/metabolismo , Unión ProteicaRESUMEN
Centipedes have a venom gland connected to a pair of forceps, which are used to arrest preys. Human victims bitten by centipedes usually manifest burning pain, paresthesia and edema, which may develop into superficial necrosis. The aim of this work was to characterize and compare toxic activities found in venoms of three species of Brazilian centipedes-Otostigmus pradoi, Cryptops iheringi and Scolopendra viridicornis. By SDS-PAGE (4-20%), important differences were noticed among venoms (between 7 and 205kDa). Few bands showed feeble caseinolytic, fibrinogenolytic and gelatinolytic activities by zymography, but strong hyaluronidase activity was observed in S. viridicornis and O. pradoi venoms. In addition, such activities could be inhibited by o-phenanthroline, indicating that these enzymes are metalloproteinases. All venoms induced nociception, edema and myotoxicity in mice, but only S. viridicornis induced mild hemorrhagic activity. No coagulant activity was detected in centipede venoms. Low phospholipase A(2) activity was observed exclusively in S. viridicornis and O. pradoi venoms, but these venoms had intense direct hemolytic activity on human erythrocytes. Cross-reactivity among venoms was observed using species-specific sera raised in rabbits. Differences were noticed among centipede venoms, but S. viridicornis is indeed the most toxic venom and thereby it could induce a more severe envenomation.
Asunto(s)
Venenos de Artrópodos/inmunología , Venenos de Artrópodos/toxicidad , Artrópodos/fisiología , Animales , Antivenenos/metabolismo , Venenos de Artrópodos/química , Reacciones Cruzadas/efectos de los fármacos , Reacciones Cruzadas/inmunología , Relación Dosis-Respuesta a Droga , Edema/inducido químicamente , Edema/patología , Eritrocitos/efectos de los fármacos , Hemólisis/efectos de los fármacos , Hemorragia/inducido químicamente , Hemorragia/patología , Humanos , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/metabolismo , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Músculo Esquelético/fisiopatología , Dolor/inducido químicamente , Dolor/fisiopatología , Dimensión del Dolor , Fenantrolinas/farmacología , Fosfolipasas A/análisis , Fosfolipasas A/metabolismo , Conejos , Piel/efectos de los fármacos , Piel/patologíaRESUMEN
Many medicinal plants have been recommended for the treatment of snakebites. The aqueous extracts prepared from the leaves of Schizolobium parahyba (a plant found in Mata Atlantica in Southeastern Brazil) were assayed for their ability to inhibit some enzymatic and biological activities induced by Bothrops pauloensis and Crotalus durissus terrificus venoms as well as by their isolated toxins neuwiedase (metalloproteinase), BnSP-7 (basic Lys49 PLA(2)) and CB (PLA(2) from crotoxin complex). Phospholipase A(2), coagulant, fibrinogenolytic, hemorrhagic and myotoxic activities induced by B. pauloensis and C. d. terrificus venoms, as well as by their isolated toxins were significantly inhibited when different amounts of S. parahyba were incubated previously with these venoms and toxins before assays. However, when S. parahyba was administered at the same route as the venoms or toxins injections, the tissue local damage, such as hemorrhage and myotoxicity was only partially inhibited. The study also evaluated the inhibitory effect of S. parahyba upon the spreading of venom proteins from the injected area into the systemic circulation. The neutralization of systemic alterations induced by i.m. injection of B. pauloensis venom was evaluated by measuring platelet and plasma fibrinogen levels which were significantly maintained when S. parahyba extract inoculation occurred at the same route after B. pauloensis venom injection. In conclusion, the observations confirmed that the aqueous extract of S. parahyba possesses potent snake venom neutralizing properties. It may be used as an alternative treatment to serum therapy and as a rich source of potential inhibitors of toxins involved in several physiopathological human and animal diseases.
Asunto(s)
Antivenenos/farmacología , Casearia/química , Fitoterapia , Extractos Vegetales/farmacología , Hojas de la Planta/química , Proteínas de Plantas/farmacología , Animales , Antivenenos/química , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/enzimología , Venenos de Crotálidos/toxicidad , Inhibidores Enzimáticos/farmacología , Fabaceae , Fibrinógeno/metabolismo , Masculino , Medicina Tradicional , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Necrosis , Fosfolipasas A/metabolismo , RosalesRESUMEN
A quantitative study of toxicity levels of the San Pedro River and its main tributaries around the city of Aguascalientes, Mexico was conducted. Our study determined individual CL(50) values for each sampling point at 3 different times of the year corresponding to the main seasons of the year in terms of the hydrological cycle (dry, low rain and high rain season). Those LC(50) values were used to calculate the acute. Toxicity Units (aTU) that allowed us to compare levels of toxicity along the San Pedro River and two of its main tributaries. The sample that showed highest toxicity was IPIVA. This is due to the large quantity of industrial discharges that receives. Its effluent was responsible for the largest contribution of toxicity to the San Pedro River over the three rounds of sampling of this study. Our study classified an important portion of the San Pedro River and two of its main tributaries in toxic, moderately toxic and lightly toxic. No portion of the river studied was free of toxicity, either acute or sublethal. This study demonstrated that in spite of the operation of several water treatment plants along the San Pedro River, for the most part, the water quality of the river is still unacceptable.
Asunto(s)
Ríos , Contaminantes Químicos del Agua/toxicidad , Animales , Ciudades , Daphnia/fisiología , Monitoreo del Ambiente , Esterasas/metabolismo , Conducta Alimentaria/efectos de los fármacos , Femenino , Dosificación Letal Mediana , México , Oxígeno/análisis , Fosfolipasas A/metabolismo , Rotíferos/fisiología , Abastecimiento de AguaRESUMEN
We have showed that a phospholipase A(2) isolated from Lachesis muta snake venom, denoted LM-PLA(2)-I, had some biological effects. Here, we examined its effects on lymphocytes. Pre-incubation of human peripheral blood lymphocytes with LM-PLA(2)-I plus phosphatidylcholine (PC) stimulated the natural killer (NK) activity. This was accompanied by DNA binding of nuclear transcription factor kappaB and the increase in PKC activity with translocation of the enzyme from the cytoplasma into the plasma membrane. These effects were reproduced when lymphocytes were pre-incubated with commercial lysophosphatidylcholine (LPC) and abolished by stausrosporin or p-bromophenacyl bromide. Evaluation of phosphorylated PKC isoforms showed that pre-incubation with LPC activated the autophosphorylation of the PKCzeta isoform. Taken together, these results confirm that the enzymatic activity of the phospholipase A(2) present in L. muta venom is for the biological activity of the snake venom, and strongly suggest that the LPC produced may be acting as a modulator of PKC isoforms.
Asunto(s)
Venenos de Crotálidos/química , Venenos de Crotálidos/enzimología , Células Asesinas Naturales/efectos de los fármacos , Lisofosfatidilcolinas/metabolismo , Fosfolipasas A/metabolismo , Proteína Quinasa C/metabolismo , Animales , Línea Celular Tumoral , Humanos , Lisofosfatidilcolinas/farmacología , Fosfatidilcolinas/metabolismo , Fosfolipasas A2 , Fosforilación , Isoformas de Proteínas , Estaurosporina/farmacología , Viperidae/metabolismoRESUMEN
The Bothrops neuwiedi (Neuwied's lancehead) species complex consists of a variety of subspecies with a wide distribution in South America. In this work, we compared the neuromuscular blockade caused by venoms from three subspecies (B. n. goyazensis, B. n. paranaensis and B. n. diporus) of this complex using chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations and investigated their phospholipase A2 (PLA2) activities and electrophoretic profiles. The order of potency of PLA2 activity was B. n. diporus>B. n. paranaensis>B. n. goyazensis. In BC preparations, B. n. goyazensis venom (50 microg/mL) was significantly (p<0.05) more active than B. n. paranaensis and B. n. diporus venoms, which did not produce a significant blockade at this time interval; after 120 min, B. n. goyazensis, B. n. paranaensis and B. n. diporus venoms (100 microg/mL) produced blockades of 57.4+/-5%, 30+/-3% and 17.4+/-7% (n=3-6 each), respectively. The three venoms inhibited contractures in response to ACh, indicating interference with postsynaptic neurotransmission. Only B. n. goyazensis and B. n. paranaensis venoms caused a long-lasting, concentration-dependent muscle contracture prior to blockade. In PND preparations, all of the venoms blocked the twitch-tension responses within 45-100 min, indicating that these preparations were more sensitive than avian preparations. There was a correlation between PLA2 activity and the time for 50% blockade in PND but not in BC preparations. SDS-PAGE showed quantitative rather than qualitative differences among the venoms. These results indicate that the venoms of the three subspecies had similar profiles of neuromuscular activity, although the relationship with PLA2 activity varied with the preparation used.
Asunto(s)
Diafragma/efectos de los fármacos , Fosfolipasas A/metabolismo , Nervio Frénico/efectos de los fármacos , Venenos de Serpiente/química , Animales , Bothrops , Pollos , Electroforesis en Gel de Poliacrilamida , Masculino , Ratones , Fosfolipasas A2 , Venenos de Serpiente/enzimología , Especificidad de la EspecieRESUMEN
BACKGROUND: Annexin 1 is a 37-kDa protein that has complex intra- and extracellular effects. To discover whether the absence of this protein alters bone development, we monitored this event in the annexin-A1 null mice in comparison with littermate wild-type controls. METHODS: Radiographic and densitometry methods were used for the assessment of bone in annexin-A1 null mice at a gross level. We used whole-skeleton staining, histological analysis, and Western blotting techniques to monitor changes at the tissue and cellular levels. RESULTS: There were no gross differences in the appendicular skeleton between the genotypes, but an anomalous development of the skull was observed in the annexin-A1 null mice. This was characterized in the newborn annexin-A1 null animals by a delayed intramembranous ossification of the skull, incomplete fusion of the interfrontal suture and palatine bone, and the presence of an abnormal suture structure. The annexin-A1 gene was shown to be active in osteocytes during this phase and COX-2 was abundantly expressed in cartilage and bone taken from annexin-A1 null mice. CONCLUSIONS: Expression of the annexin-A1 gene is important for the normal development of the skull in mice, possibly through the regulation of osteoblast differentiation and a secondary effect on the expression of components of the cPLA2-COX-2 system.
Asunto(s)
Anexina A1/genética , Huesos/anomalías , Anomalías Craneofaciales/genética , Expresión Génica/fisiología , Animales , Animales Recién Nacidos , Densidad Ósea , Desarrollo Óseo/genética , Anomalías Craneofaciales/metabolismo , Anomalías Craneofaciales/patología , Ciclooxigenasa 2/metabolismo , Femenino , Homocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteogénesis , Fosfolipasas A/metabolismoRESUMEN
Scanning alanine mutagenesis has been used to study the structural determinants of several activities of bothropstoxin-I (BthTx-I), a lysine 49 Phospholipases A(2) from the venom of Bothrops jararacussu. A total of 31 mutants were generated in the interfacial recognition site and C-terminal loop regions of the protein. The effects of mutagenesis on the in vivo myotoxic activity, the cytolytic activity against cultured C2C12 myoblasts, the bactericidal activity, and the Ca(2+)-independent membrane damaging activity against liposome membranes were compared. Residues 116-119 and 122-125 in the C-terminal loop region are structural determinants for these activities, indicating that membrane permeabilization by the BthTx-I is an important general property in all the measured effects. The structural determinants of myotoxicity and myoblast membrane permeabilization are highly correlated, demonstrating that cultured C2C12 myoblasts are a good model for the myotoxic effect. However, comparison of the structural determinants for all activities revealed several differences in the structural determinants between the effects against myoblast and bacterial membranes, and further differences when compared to the liposome membrane damaging effect. These membrane dependent effects are interpreted to be the consequence of differences in the activation of the membrane bound form of the protein on biological and artificial membranes.