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@#Malaria, a mosquito-borne disease, is caused by protozoa of the genus Plasmodium and constitutes a serious public health problem. Because current insecticides used to control malaria face resistance due to continuous use, new alternatives are prompted. Considering this context, and the insecticidal potential of vertebrate venoms/secretions, crude and methanolic extracts from two frog species were tested as larvicides against Anopheles darlingi. Skin secretions of Rhinella marina and Rhaebo guttatus were obtained by manual stimulation. Then, methanol was added to obtain steroidal fractions from both venoms. Mosquitos were captured in suburban areas of Porto Velho and An. darlingi females were later fed with blood and stimulated to oviposit. The larvae were fed with fish food until the 3rd and 4th instars. For the larvicidal assays, crude secretions and methanolic fractions of both frog species were evaluated, and larvae mortality was recorded after 48 hours. Crude extracts and steroidal fractions from both species had larvicidal effects, with an LC50 of 127.5 and 133 ppm for the crude extract and steroidal fraction of R. marina, and an LC50 of 37.5 and 35.8 ppm for the crude extract and steroidal secretion of R. guttatus, respectively. The present work reports for the first time the larvicidal effects of the skin secretions from bufonid species occurring in the western Amazon region. Further studies should be carried out to investigate the purified components responsible for the observed activity.
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Vinte e seis cães braquicefálicos portadores de estenose de narina, 22 machos e quatro fêmeas, foram submetidos à rinoplastia bilateral. Dezesseis cães eram Buldogues Franceses; dois, Buldogues Ingleses; seis, Pugs; e dois, Shih Tzus, com idade variando de seis meses a seis anos. Foram efetuadas coletas de sangue arterial para análises hemogasométricas no pré-operatório e 30 dias após a cirurgia. Para cada avaliação, foi obtida uma amostra de 0,5mL de sangue coletado da artéria femoral, em seringa plástica heparinizada. Em seguida, procedeu-se à avaliação hemogasométrica em analisador de gases sanguíneos (I-stat-Abbot®). Os resultados da hemogasometria pré e pós-rinoplastia mostraram uma redução nos valores médios de pCO2, TCO2 , HCO3- e BEecf, hematócrito e hemoglobina, e aumento de pH, pO2 e SO2, indicando melhora na condição ventilatória dos animais após a correção cirúrgica da estenose de narina. Isso posto, conclui-se que a hemogasometria arterial é um exame importante no diagnóstico da síndrome respiratória dos cães braquicefálicos, e extremamente útil no acompanhamento da resposta do paciente ao tratamento. A rinoplastia mostrou-se eficaz no tratamento da síndrome respiratória, promovendo melhora nos parâmetros hemogasométricos que indicam acidose respiratória secundária à obstrução das vias aéreas, comum nas raças braquicefálicas.(AU)
Twenty-six brachycephalic dogs with nostril stenosis, 22 males and four females, underwent bilateral rhinoplasty. Sixteen dogs were French Bulldogs; two, English Bulldogs; Six, Pugs; and two, Shih tzus, ranging in age from six months to six years. Blood samples were collected for hemogasometric analysis in the preoperative period and 30 days after surgery. For each evaluation, a 0.5ml sample of blood collected from the femoral artery was obtained in a heparinized plastic syringe. Hemogasometric evaluation was then performed on a blood gas analyzer (I-stat-Abbot®). The results of hemogasometry before and after rhinoplasty showed a reduction in the mean values of pCO2, TCO2, HCO3- and BEecf, hematocrit and hemoglobin, and an increase in pH, pO2 and SO2, indicating an improvement in the ventilatory condition of the animals after surgical correction of Nostril stenosis. Therefore, it is concluded that arterial hemogasometry is an important diagnostic tool for the diagnosis of brachycephalic respiratory syndrome and is extremely useful in monitoring the patient's response to treatment. Rhinoplasty was effective in the treatment of respiratory syndrome, promoting improvement in hemogasometric parameters that indicate respiratory acidosis secondary to airway obstruction, common in the brachycephalic races.(AU)
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Animales , Perros , Acidosis Respiratoria , Constricción Patológica , Perros/sangreRESUMEN
Undernutrition represents a major public health challenge for middle- and low-income countries. This study aimed to evaluate whether a multideficient Northeast Brazil regional basic diet (RBD) induces acute morphological and functional changes in the ileum of mice. Swiss mice (∼25 g) were allocated into two groups: i) control mice were fed a standard diet and II) undernourished mice were fed the RBD. After 7 days, mice were killed and the ileum collected for evaluation of electrophysiological parameters (Ussing chambers), transcription (RT-qPCR) and protein expression (western blotting) of intestinal transporters and tight junctions. Body weight gain was significantly decreased in the undernourished group, which also showed decreased crypt depth but no alterations in villus height. Electrophysiology measurements showed a reduced basal short circuit current (Isc) in the undernourished group, with no differences in transepithelial resistance. Specific substrate-evoked Isc related to affinity and efficacy (glutamine and alanyl-glutamine) were not different between groups, except for the maximum Isc (efficacy) induced by glucose. Transcription of Sglt1 and Pept1 was significantly higher in the undernourished group, while SN-2 transcription was decreased. No changes were found in transcription of CAT-1 and CFTR, while claudin-2 and occludin transcriptions were significantly increased in the undernourished group. Despite mRNA changes, SGLT-1, PEPT-1, claudin-2 and occludin protein expression showed no difference between groups. These results demonstrate early effects of the RBD on mice, which include reduced body weight and crypt depth in the absence of significant alterations to villus morphology, intestinal transporters and tight junction expression.
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Animales , Masculino , Conejos , Desnutrición/fisiopatología , Desnutrición/metabolismo , Crecimiento/fisiología , Íleon/anatomía & histología , Íleon/metabolismo , Fenómenos Fisiológicos Nutricionales de los Animales , Factores de Tiempo , Peso Corporal , Ingestión de Energía/fisiología , ARN Mensajero , Immunoblotting , Enfermedad Aguda , Transporte Iónico/fisiología , Desnutrición/complicaciones , Modelos Animales de Enfermedad , Absorción Intestinal/fisiologíaRESUMEN
Cell-free antigens (CFAg) derived from Paracoccidioides brasiliensis have typically been used in immunodiffusion reactions for serodiagnosis or therapeutic follow-up of paracoccidioidomycosis patients. Thus, we investigated the usefulness of CFAg obtained from cultures at different ages, to evaluate cellular immunity by the footpad test, in experimental murine paracoccidioidomycosis. Male mice infected with P. brasiliensis 265 strain were challenged in the footpad with CFAg obtained from four- (4d CFAg) or 11-day-old cultures (11d CFAg). The increase in footpad swelling provoked by 4d CFAg and 11d CFAg was similar and showed significant difference in relation to control groups. However, the infiltrate pattern was strikingly different: 4d CFAg induced a predominant mononuclear infiltrate whereas 11d CFAg provoked a predominant polymophonuclear infiltrate. These different inflammatory patterns were associated with distinct electrophoretic characteristics. By comparison with 11d CFAg, 4d CFAg showed more numerous and intense bands, including a strong one of 43 kDa (gp43). These results suggest that CFAg derived from Pb 265 isolate can be used as a reagent to evaluate cellular immunity; however, the culture's age is critical because only young cultures are able to induce a typical mononuclear infiltrate. The efficacy of this new paracoccidioidin to assay the cellular immunity in infections caused by other P. brasiliensis isolates is under investigation.
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Animales , Masculino , Ratones , Hipersensibilidad Tardía , Paracoccidioides , ParacoccidioidomicosisRESUMEN
Gyroxin, a thrombin-like enzyme isolated from Crotalus durissus terrificus venom and capable of converting fibrinogen into fibrin, presents coagulant and neurotoxic activities. The aim of the present study was to evaluate such coagulant and toxic properties. Gyroxin was isolated using only two chromatographic steps - namely gel filtration (Sephadex G-75) and affinity (Benzamidine Sepharose 6B) - resulting in a sample of high purity, as evaluated by RP-HPLC C2/C18 and electrophoretic analysis that showed a molecular mass of 30 kDa. Gyroxin hydrolyzed specific chromogenic substrates, which caused it to be classified as a serine proteinase and thrombin-like enzyme. It was stable from pH 5.5 to 8.5 and inhibited by Mn²+, Cu²+, PMSF and benzamidine. Human plasma coagulation was more efficient at pH 6.0. An in vivo toxicity test showed that only behavioral alterations occurred, with no barrel rotation. Gyroxin was not able to block neuromuscular contraction in vitro, which suggests that its action, at the studied concentrations, has no effect on the peripheral nervous system.
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Animales , Ratas , Venenos de Crotálidos , Trombina/aislamiento & purificación , Trombina/toxicidadRESUMEN
Most of the snakebites recorded in Brazil are caused by the Bothrops genus. Given that the local tissue damage caused by this genus cannot be treated by antivenom therapy, numerous studies are focusing on supplementary alternatives, such as the use of medicinal plants. Serjania erecta has already demonstrated anti-inflammatory, antiseptic and healing properties. In the current study, the aerial parts of S. erecta were extracted with methanol, then submitted to chromatographic fractionation on a Sephadex LH20 column and eluted with methanol, which resulted in four main fractions. The crude extract and fractions neutralized the toxic activities of Bothrops jararacussu snake venom and isolated myotoxins (BthTX-I and II). Results showed that phospholipase A2, fibrinogenolytic, myotoxic and hemorrhagic activities were inhibited by the extract. Moreover, the myotoxic and edematous activities induced by BthTX-I, and phospholipase A2 activity induced by BthTX-II, were inhibited by the extract of S. erecta and its fraction. The clotting time on bovine plasma was significantly prolonged by the inhibitory action of fractions SF3 and SF4. This extract is a promising source of natural inhibitors, such as flavonoids and tannins, which act by forming complexes with metal ions and proteins, inhibiting the action of serineproteases, metalloproteases and phospholipases A2.
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Animales , Masculino , Ratones , Bothrops , Extractos Vegetales/antagonistas & inhibidores , Plantas Medicinales , Venenos de Crotálidos/toxicidad , AntivenenosRESUMEN
Envenoming by Bothrops snakes is the most serious type of envenoming from the medical and economic point of view in Central America. Bothrops asper is responsible for 90% of the snakebites registered in Panamá every year. Despite its medical and economic relevance, only the venom of Costa Rican and Guatemalan populations of this species has been studied to some detail, and there is very little information on intraspecies variability in venom composition and toxicity. In this study the crude venom of B. asper from Panamá was characterized and its pharmacological and biochemistry activities were investigated with standard laboratory assays. Furthermore, we described the isolation, functional and structural characterization of four basic phospholipases A2, namely MTX-I, MTX-II, MTX-III, MTX-IV, and a new acid phospholipase A2 called Basp-I-PLA2. The proteins were isolated from the crude venom by a combination of two chromatographic steps, using ion-exchange chromatography on CM-Sepharose (0.05 M NH4HCO3 pH 8.1 buffer), and hydrophobic chromatography on Phenyl-Sepharose (0.05 M Tris-HCl pH 7.4), followed by concentration gradient from 4 to 0 M NaCl at 25°C in the same buffer. Analyses of phospholipids hydrolyzed by these enzymes have shown that all phospholipases belong to type A2. The acidic isoform demonstrated more catalytic activity than basic PLA2s. This enzyme was more active on substrates such as phosphotidylcholine and phosphatidylglycerol.(AU)
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Animales , Venenos de Serpiente/aislamiento & purificación , Bothrops , Fosfolipasas A2 , MiotoxicidadRESUMEN
The prominent myotoxic effects induced by Bothrops jararacussu crude venom are due, in part, to its polycationic myotoxins, BthTX-I and BthTX-II. Both myotoxins have a phospholipase A2 structure: BthTX-II is an active enzyme Asp-49 PLA2, while BthTX-I is a Lys-49 PLA2 devoid of enzymatic activity. In this study, the effect of low-level laser therapy (LLLT), 685 nm laser at a dose of 4.2 J/cm2 on edema formation, leukocyte influx and myonecrosis caused by BthTX-I and BthTX-II, isolated from Bothrops jararacussu snake venom, was analyzed. BthTX-I and BthTX-II caused a significant edema formation, a prominent leukocyte infiltrate composed predominantly by neutrophils and myonecrosis in envenomed gastrocnemius muscle. LLLT significantly reduced the edema formation, neutrophil accumulation and myonecrosis induced by both myotoxins 24 hours after the injection. LLLT reduced the myonecrosis caused by BthTX-I and BthTX-II, respectively, by 60 and 43 percent; the edema formation, by 41 and 60.7 percent; and the leukocyte influx, by 57.5 and 51.6 percent. In conclusion, LLLT significantly reduced the effect of these snake toxins on the inflammatory response and myonecrosis. These results suggest that LLLT should be considered a potential therapeutic approach for treatment of local effects of Bothrops species venom.
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Animales , Masculino , Ratas , Bothrops , Venenos de Crotálidos , Edema/inducido químicamente , Terapia por Luz de Baja Intensidad/métodosRESUMEN
Snakebites comprise a serious health problem in several countries due to their global incidence, which exceeds 2.5 million per year, and the elevated number of victim fatalities. To counteract envenomations, antivenoms have been used regularly for more than a century. Apart from side effects including anaphylactic reactions, antivenoms are not able to efficiently neutralize local tissue damage, which contributes to increasing the severity and morbidity observed in patients. This fact, in turn, may be responsible for economic hardship, particularly in rural populations of developing countries. In the present work, we evaluated the antiophidian properties of 12 Brazilian plant extracts against the hemolytic, coagulant, hemorrhagic and proteolytic effects of Lachesis muta venom. Taken together, our data revealed that most of these aqueous products were capable of inhibiting those activities at different levels, except for Sapindus saponaria extract. In contrast, Stryphnodendron barbatiman extract completely neutralized all the analyzed biological activities. Thus, we may conclude that Brazilian flora may also be useful against L. muta accidents.
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Animales , Ratas , Antivenenos , Lachesis muta , Extractos Vegetales , FitoterapiaRESUMEN
This article reports the anti-inflammatory effect of Blutaparon portulacoides (B. portulacoides), specifically the ethanolic extract of its aerial parts, on the edema formation and leukocyte influx caused by Bothrops jararacussu (B. jararacussu) snake venom and Bothropstoxin-I and II (BthTX-I and II) isolated from this venom as an alternative treatment for Bothrops snakebites. The anti-inflammatory effect of B. portulacoides ethanolic extract was compared with an animal group pretreated with dexamethasone. B. portulacoides ethanolic extract significantly inhibited paw edema induced by B. jararacussu venom and by BthTX-I and II. Also, results demonstrated that the extract caused a reduction of the leukocyte influx induced by BthTX-I. However, the extract was not capable of inhibiting the leukocyte influx induced by the venom and by BthTX-II. In conclusion, these results suggest that the ethanolic extract of this plant possess components able to inhibit or inactivate toxins present in B. jararacussu venom, including its myotoxins, responsible for the edema formation. However, the leukocyte migration caused by the venom and BthTX-II was not inhibited by the plant, probably due to the different mechanisms involved in the edema formation and leukocyte influx. This is the first report of B. portulacoides extract as anti-inflammatory against snake venoms and isolated toxins.(AU)
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Animales , Mordeduras de Serpientes , Venenos de Serpiente , Bothrops , Antiinflamatorios/administración & dosificaciónRESUMEN
Two presynaptic phospholipases A2 (PLA2), neuwieditoxin-I (NeuTX-I) and neuwieditoxin-II (NeuTX-II), were isolated from the venom of Bothrops neuwiedi pauloensis (BNP). The venom was fractionated using molecular exclusion HPLC (Protein-Pak 300SW column), followed by reverse phase HPLC (æBondapak C18 column). Tricine-SDS-PAGE in the presence or absence of dithiothreitol showed that NeuTX-I and NeuTX-II had a molecular mass of approximately 14 kDa and 28kDa, respectively. At 10æg/ml, both toxins produced complete neuromuscular blockade in indirectly stimulated chick biventer cervicis isolated preparation without inhibiting the response to acetylcholine, but NeuTX-II reduced the response to KCl by 67.0±8.0 percent (n=3; p<0.05). NeuTX-I and NeuTX-II are probably responsible for the presynaptic neurotoxicity of BNP venom in vitro. In fact, using loose patch clamp technique for mouse phrenic nerve-diaphragm preparation, NeuTX-I produced a calcium-dependent blockade of acetylcholine release and caused appearance of giant miniature end-plate potentials (mepps), indicating a pure presynaptic action. The N-terminal sequence of NeuTX-I was DLVQFGQMILKVAGRSLPKSYGAYGCYCGWGGRGK (71 percent homology with bothropstoxin-II and 54 percent homology with caudoxin) and that of NeuTX-II was SLFEFAKMILEETKRLPFPYYGAYGCYCGWGGQGQPKDAT (92 percent homology with Basp-III and 62 percent homology with crotoxin PLA2). The fact that NeuTX-I has Q-4 (Gln-4) and both toxins have F-5 (Phe-5) and Y-28 (Tyr-28) strongly suggests that NeuTX-I and NeuTX-II are Asp49 PLA2.
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Animales , Bothrops/metabolismo , Venenos de Crotálidos , Fosfolipasas A/química , Neurotoxinas/envenenamientoRESUMEN
This work succinctly describes the professional and scientific life of Dr. José R. Giglio, one of the most outstanding Brazilian researchers in the field of Toxinology. During his long and successful career, he has made major contributions, especially in elucidating the function, structure, and mechanisms of action of animal venom proteins (from snakes, scorpions and spiders) as well as the characterization of antibodies and several inhibitors of venoms and toxins. We present here a brief history of Dr. Giglios personal and professional life, also reporting some of his numerous published scientific articles on venoms from snakes (Bothrops, Crotalus, and other genera), scorpions (Tityus sp), spiders (Phoneutria sp), their isolated toxins and natural inhibitors. Thus, this work is a tribute to Dr. Giglio in his 73rd birthday, having devoted 48 years of his life studying animal venoms, an effort that has continued even after his formal retirement from university duties.
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Animales Ponzoñosos , Toxicología/historia , Venenos de Escorpión , Venenos de Araña , Venenos de Serpiente/antagonistas & inhibidoresRESUMEN
Two fibrinogenolytic enzymes, Bothrops alternatus metalloprotease isoform (BaltMP)-I and II, were purified from Bothrops alternatus venom using Diethylaminoethyl (DEAE) Sephacel, Sephadex G-75 and Heparin-Agarose column chromatography. Purified BaltMP-I and II ran as single protein bands on analytical polyacrylamide gel electrophoresis and showed molecular weights of 29000 and 36000, respectively, under reducing conditions in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). BaltMP-II, but not BaltMP-I, displayed blood-clotting activity in bovine plasma, which was about 10-fold higher than that of the crude venom. Both enzymes were proteolytically active against bovine fibrinogen as substrate. When fibrinogen and each enzyme were incubated at 37°C, at a ratio of 1:100 (w/w), BaltMP-II cleaved preferentially the Aalpha -chain and more slowly the Bbeta -chain. The action of BaltMP-I was similar, but lower. None of the proteases degraded the gamma-chain of fibrinogen. The fibrinogenolytic activity of the enzymes was inhibited by 1,10-phenanthroline, suggesting they are metalloproteases. Since both enzymes were found to cause defibrinogenation when intraperitoneally (i.p.) administered to mice, they can be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.(AU)
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Animales , Fibrinógeno/aislamiento & purificación , Bothrops , Venenos de Crotálidos/aislamiento & purificación , Metaloproteasas , Trombosis , Isoformas de ProteínasRESUMEN
This paper describes a brief study on the crotoxin mechanism of action, regarding the transport of GABA and L-glutamate in rats cortico-cerebral synaptosomes and in heterologous systems, such as COS-7 cells expressing gabaergic transporters, and C6 glioma cells and Xenopus oocytes expressing glutamatergic transporters. Crotoxin concentrations over 1 µM caused an inhibitory effect of ³H-L-glutamate and ³H-GABA, and reversibly inhibited L-glutamate uptake by C6 glioma cells. When COS-7 cells were assayed, no inhibition of the ³H-GABA transport could be evidenced. Crotoxin kept its inhibitory effect on neurotransmitters uptake even when Ca2+ ions were removed from the medium, therefore, independently of its PLA2 activity. In addition, high concentrations (2 mM) of BPB did not avoid the action of crotoxin on the neurotransmitters uptake. Crotoxin also inhibited ³H-L-glutamate, independently on Na+ channel blockade by TTX. In addition, an evaluation of the lactic dehydrogenase activity indicated that uptake inhibition does not involve a hydrolytic action of crotoxin upon the membrane. We may also suggest that crotoxin acts, at least partially, altering the electrogenic equilibrium, as evidenced by confocal microscopy, when a fluorescent probe was used to verify cell permeability on C6 glioma cells in presence of crotoxin.
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Animales , Masculino , Ratas , GABAérgicos , Crotoxina , Glutamatos , Neurotoxinas , Venenos de Crotálidos/farmacología , Sistema NerviosoRESUMEN
Beta-glucan, one of the major cell wall components of Saccharomyces cerevisiae, has been found to enhance immune functions. This study investigated in vivo and in vitro effects of beta-glucan on lymphoproliferation and interferon-gamma (IFN-gamma) production by splenic cells from C57BL/6 female mice. All experiments were performed with particulate beta-glucan derived from S. cerevisiae. Data demonstrated that both, i.p administration of particulate beta-glucan (20 or 100 µg/animal) and in vitro stimulation of splenic cells (20 or 100 µg/ml of culture) decreased lymphoproliferation and IFN-gamma production induced by concanavalin A. These results suggest that beta-glucan can trigger a down-modulatory effect regulating a deleterious immune system hyperactivity in the presence of a strong stimulus.
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Animales , Femenino , Ratones , Adyuvantes Inmunológicos , Glucanos , Interferón gamma , Activación de Linfocitos , Saccharomyces cerevisiae , Bazo , Concanavalina A , Glucanos , Interferón gamma , Activación de Linfocitos , Ratones Endogámicos C57BL , BazoRESUMEN
Snake venoms frequently vary in composition. In this work, we compared the neurotoxic and myotoxic activities of 16 lots of Bothrops neuwiedii venoms from different regions of Brazil, using chick biventer cervicis preparations. The neuromuscular blockade varied from 2 per cent to 100 per cent after 120 min incubation with venoms (50µg/ml). In all cases, this blockade was irreversible and concentration-dependent; at low concentrations (10-20 µg/ml), 15 of the 16 venom lots failed to abolish responses to acetylcholine (110µM), but blocked responses to KCI (13.4mM), and induced contracture. At 5-20µg/ml, the most active venom totally blocked twitch-tension without affecting responses to acetylcholine and KCI. Polyacrylamine gel electrophoresis for basic proteins showed that the most active samples contained a band that was absent in the less active venoms. These results indicate that there may be considerable intraspecific variation in the neurotoxic activity of B. ineuwiedii venoms, whereas myotoxic activity is less variable.
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Animales , Masculino , Bothrops , Brasil , Pollos , Miotonía , Sistema Nervioso , Neurotoxinas , Venenos de Crotálidos/efectos adversos , Venenos de Crotálidos/toxicidad , Acetilcolina , Contractura , Bloqueo NeuromuscularRESUMEN
Propolis has been the subject of several recent studies, with the aim of elucidating its biological and pharmacological properties. Propolis has a well-known antimicrobial activity as well as antioxidant, antitumoral, antiinflammatory, and regenerative properties, but literature about its effects on the immunes response in scarce. The goal of this work was to evaluate the propolis effect on macrophage activation by oxygen (H2O2) and nitrogen (NO) metabolite determination. Propolis was produced by africanized honeybees and hydroalcoholic solutions were prepared at different concentrations. Peritoneal macrophages were obtained from male BALB/c mice and culture cells were stimulated in vitro with propolis or interferon-gamma (IFN-gamma). In the in vivo assay, the animals were sacrificed after propolis treatment and cells were stimulated with IFN-gamma. We also investigated the co-stimulant action of propolis associated with IFN-gamma on macrophages. The results show that propolis induces a discreet elevation in H2O2 release and a mild inhibition of NO generation, depending on concentration. Propolis had no co-stimulant activity, diminishing IFN-gamma action on H2O2 and NO production. Data suggest that propolis acts on host non-specific immunity by macrophage activation.
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Animales , Masculino , Ratas , Activación de Macrófagos , Nitrógeno/metabolismo , Oxígeno/metabolismo , Peróxido de Hidrógeno/metabolismo , Própolis/farmacología , Abejas , Interferón gamma/metabolismo , Macrófagos PeritonealesRESUMEN
Polyacrylamide gel electrophoresis (PAGE) for basic proteins may be a useful toll for the characterization of whole snake venoms and for the taxonomic classification of snakes of the Elapidae and Viperidae families. However, due to the close proximity of PAGE was not able to provide an efficient differentiation. This article reports the electrophoretic analysis of several venoms from the genera Micrurus, Bothrops, Bothriopsis, Crotalus and Lachesis and shows a typical and distinctive electrophoretic profile for each species, with intraspecific and geographic variation. Even in cases in which extreme morphological similarities were present, such as between B. jararacussu and B. pirajai ("Bahia jararacussu"), differentiation could be evidenced by PAGE. This simple and sensitive procedure may be applied to similar cases involving basic toxins.
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Animales , Elapidae/clasificación , Electroforesis en Gel de Poliacrilamida , Venenos de Víboras/química , Venenos Elapídicos/química , Viperidae/clasificación , BrasilRESUMEN
Guanylin is an endogenous peptide synthesized by several mammalian species that mimics the effects of a thermostable enterotoxin of Escherichia coli (STa: NTFYCCELCCNPACAGCY) in the gut. We have cloned a lysine-1 derivative of rat guanylin (Lys-1-NTCEICAYAACTGC) and tested its effects on ileal tissue membranes in Ussing chambers and in the isolated perfused rat kidney. Rabbit ileal mucosa membranes were mounted into a Ussing chamber and the effects of Lys-1 guanylin (Lys-1 G) and STa enterotoxin peptide on chloride secretion were determined by changes in short-circuit current (Isc). Lys-1 G (10 to 100 nM) showed a dose-dependent effect on chloride secretion with a maximal response estimated to be 52 microA/cm2. Lys-1 G mimics the effect of STa peptide, but the enterotoxin elicited a greater maximal effect of 120 microA/cm2 (p<0.01). Lys-1 G (2.5 microg/ml) promoted an increase in both urine flow (from 0.13 +/- 0.07 to 0.40 +/- 0.01 ml g(-1) min(-1), N = 4; P<0.05) and glomerular filtration rate (from 0.68 +/- 0.02 to 0.85 0.00 ml g(-1) min(-1), N = 4; P<0.01) in the isolated perfused kidney and a reduction of the fractional reabsorption of sodium (from 76.0 +/- 0.03 to 59.5 +/- 0.85 percent, N = 4; P<0.01). These maximal effects were accompanied by intense natriuretic effect observed 30 and 60 min after drug administration. The Lys-1 G analog similar to STa enterotoxin elicited intestinal chloride secretion and a natriuretic effect. These data demonstrate that the cloned peptide analog retains the biological activity of the native hormone and presents activity similar to STa. The properties of Lys-1 G resemble those of a factor formed during perfusion of the hypoxic rabbit kidney and named by us factor natriureticus similis (FNS).
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Animales , Masculino , Femenino , Ratas , Conejos , Riñón/efectos de los fármacos , Lisina/análogos & derivados , Natriuresis/efectos de los fármacos , Secreciones Intestinales , Riñón/fisiología , Sodio/metabolismoRESUMEN
To investigate the effect of substrates during oral rehydration therapy, we studied intestinal cation cotransport (ICC) with glutamine (Gln) alanine (Ala) and glucose (Glu). The specific aims were to determine the biological effects of these three different cotransport systems on intestinal function. Isolated rabbit ileal mucosa preparations mounted in Ussing chambers were studied. ICC was determined by measuring short-circuit current (Isc) and potential difference (PD) while monitoring toissue resistance (TR). The data are reported as the mean ñ SEM of 4-6 experiments for each amino acid concentration. Increasing concentrations of Gln (10-5 to 10-2 M), Ala (10-5 to 10-1 M) and Glu (10-5 to 10-2 M) caused a significant (P<0.05) increase in ICC. Glin (30 mM) and Ala (0.1 M) had a maximal effects (Em (Glin)=100% anmd Em (Ala)=66%, P<0.05) which was higher than that obtained with 30 mM Glu (Em (Glu)=35%). When sodium was replaced with choline on the mucosal side. Ringer solution completeley abolished the response with Gln, Ala and Glu. The presence of all three substrates (10-2 M gln, 10-1 M Ala and 10-2 M Glu) in Ringer solution on the mucosal side caused a significant increase in ICC ( increase of short circuit current = III ñ 43 uA, P<0.05). These results demonstrate that Glin, Ala and Glu each increased sodium-dependent cation cotransport, and that sodium-dependent intestinal cation cotransport was higher with Gln than with Ala or Glu