Preparation of purified proteins from fresh Pheretima and their inhibitory effect against pulmonary fibrosis in mice / 南方医科大学学报

OBJECTIVE@#To develop a convenient method for rapid purification of fresh Pheretima proteins and assess the inhibitory effect of these proteins against pulmonary fibrosis.@*METHODS@#The crude extract of fresh Pheretima was obtained by freeze-drying method and then purified by size exclusion chromatography. The composition of the purified proteins was analyzed by mass spectrometry. MRC-5 cells were treated with 5 ng/mL TGF-β1 alone (model group) or in combination with SB431542 (2 μmol/L) or the purified proteins (13.125 μg/mL), and the cytotoxicity of purified proteins and their inhibitory effects on cell proliferation were detected with CCK8 assay. Flow cytometry was used to detect the changes in cell apoptosis, and the cellular expressions of α-SMA, Vimentin, E-cadherin, collagen I, Smad2/3 and P-Smad2/3 were detected using RT-PCR and Western blotting. In the animal experiment, adult male C57BL/6 mice were subjected to intratracheal instillation of bleomycin followed by treatment with the purified proteins (5 mg/mL) for 21 days, after which HE and Masson staining was used to observe the pathological changes in the lung tissue of the mice.@*RESULTS@#We successfully obtained purified proteins from fresh Pheretima protein by size exclusion chromatography. Treatment with the purified proteins significantly inhibited TGF-β1-induced proliferation of MRC-5 cells (P < 0.01), reduced the cellular expressions of α-SMA, Vimentin and collagen I (P < 0.001 or P < 0.01), increased the expression of E-cadherin (P < 0.01), and inhibited the expressions of Smad2/3 and P-Smad2/3 (P < 0.001 or P < 0.01). In male C57BL/6 mice models of bleomycin-induced pulmonary fibrosis, treatment with the purified proteins obviously reduced the number of inflammatory cells and fibrotic area in the lungs.@*CONCLUSION@#The purified proteins from fresh Pheretima obtained by size exclusion chromatography can inhibit pulmonary fibrosis in mice by regulating the TGF-β/ Smad pathway.

Avaliação da dissipação do inseticida malation utilizado em nebulização a ultrabaixo volume no controle da dengue: avaliação ecotoxicológica e de risco ambiental / Evaluation of malathion insecticides dissipation used in ultra-low volume nebulization in dengue control ecotoxicological assessment and environmental risk

A dengue é um dos principais problemas de Saúde Pública da atualidade. É transmitida por mosquitos do gênero Aedes, sendo a espécie Aedes aegypti o vetor responsável pela transmissão no Brasil. O principal alvo de controle da doença é seu vetor, sendo o controle químico amplamente utilizado em todas as regiões atingidas. Como forma de prevenção ou em períodos de epidemia, torna-se necessária a eliminação dos mosquitos adultos, sendo indicada a nebulização a ultrabaixo volume (UBV) a frio do organofosforado malation por meio do uso de equipamentos pulverizadores acoplados a veículos. Este inseticida utilizado pode sofrer desvios durante e após a aplicação a UBV, caracterizando-se como um potencial agente de desequilíbrio ecológico, podendo atingir e gerar efeitos danosos em organismos não alvos aquáticos e terrestres. Este trabalho teve como objetivo desenvolver e validar um método de análise para o malation em cromatografia líquida de alta eficiência e obter recuperação do inseticida em níveis aceitáveis em água e solo, avaliar seu período de dissipação e tempo de meia vida (t1/2) nestas matrizes e classificá-lo quanto à toxicidade aguda e ao risco ambiental de acordo com diferentes autores, para o microcustáceo Daphnia magna, para a minhoca Eisenia foetida, para o peixe mato grosso (Hyphessobrycon eques) e para a macrófita Lemna minor. O método foi considerado adequado para análise do malation. A recuperação obtida para água foi de 97 por cento e para solo, 96 por cento...

Dengue is one of the major public health problems nowadays. It is transmitted by mosquitoes of the genus Aedes, and the Aedes aegypti specie is the vector responsible for transmission in Brazil. The main target to control the disease is its vector, and the chemical control is widely used in all affected regions. As a preventive measure or in epidemic periods, it becomes necessary to eliminate adult mosquitoes, being indicated the ultralow volume nebulization of malathion organophosphate in cold way through the use of sprinklers attached to vehicles. This used insecticide can suffer deviation during and after application ULV, characterizing itself as a potential agent of environmental imbalance, with the possibility of reach and generate harmful effects on aquatic and terrestrial non-target organisms. This work had the objective to develop and validate a method of analysis for malathion in high performance liquid chromatography and obtain recovery of insecticide in acceptable levels in water and soil, evaluate its dissipation period and half-life time in these matrices and classify it on acute toxicity and environmental risk according to different authors, to the microcrustacean Daphnia magna, to the earthworm Eisenia foetida, to the fish Hyphessobrycon eques and to the macrophyte Lemna minor. The method was considered appropriate for analysis of malathion. The obtained recovery for water was 97 per cent and for soil, 96 per cent . Toxic effects resulting from exposure to...

Toxicity of imidacloprid [nicotinoid] against earthworm, pheretima posthuma with reference to its effects on protein

Imidaclorprid was studied for its effects on earthworm [P. posthumd]. The organism was reared in laboratory at 25 °C with relative humidity 80%, in whole moist soil with compost / manure as food medium contained in wide mouth jars covered with muslin cloth. Imidacloprid [Nicotinoid] was used as test material. Earthworms were exposed to the under test compound through contact method cum feeding method. Average mortalities of imidacloprid were found to be 20, 40, 60, 70 and 80%, against 0.04, 0.08, 0.16, 0.32, and 0.64 ppm respectively and LD50 was determined statistically and found to be 0.11 ppm. Effect of this pesticide on total protein content was determined in head, clitellum and abdominal regions of treated earthworm P. posthuma. Protein was found to be 21.6 mg/ml in head region, in clitellum region it was found to be 36.6, and 30.9 mg/ml in abdominal region respectively, whereas in the control batches it was 27.7, 26.1 and 30.3 mg/ml in head, clitellum and abdominal region respectively. This pattern indicates an increase in protein contents in the clitellum region, a decrease in head region and almost unaffected protein contents was found in the abdominal regions under the effects of imidacloprid on earthworms

Efecto de sedimentos naturales enriquecidos con zinc, en modelos aislados y en microcosmos, sobre tres especies de invertebrados bentónicos / Effect of zinc-enriched sediments, in open and isolated systems, on three species of benthonic invertebrates

Availability of toxics in aquatic bodies is limited by the physicochemical characteristics of sediments and water, as well as by the interactions between the different xenobiotics and inhabits species. The aim of this work was to relate the effect produced by zinc (Zn) spiked in sediments of the Ignacio Ramirez dam (PIR), in isolated and microcosms models, on ATP concentration of three benthic organisms with the metal biodisponibility. The selected species were a crustacean, an annelid and a mollusk: Hyalella azteca (Amphipoda: Hyalellidae), Limnodrilus hoffmeisteri (Oligochaeta: Tubificidae) and Stagnicola attenuata (Basommatophora: Lymnaeidae), species that are found at high proportions in the reservoir and use different spaces in the benthos. Samples of sediments and organisms were collected from the PIR during the dry season (February of 1999). Metal concentration (Zn, Fe, Cu and Ni), pH, texture, particle size, total nitrogen and organic matter were determined in sediments. Sublethal studies were carried out using two types of static systems (organisms isolated and in microcosms). Both models contained PIR sediments enriched with Zn (nominal concentration of 0.8129 mg/kg) and synthetic water in a proportion of 1:4. The test organisms were added to the systems once the equilibrium was reached (2 hr) considering the biomass quantity with respect to volume (1.0 g of organism by each 100 ml of water:sediment). After 0, 12, 24, 36, 48 and 72 hr of exposure, samples of sediment and hydrobionts were taken, and Zn content was quantified by atomic absorption. ATP concentration was also determined in organisms. The effect produced by natural sediments spiked with Zn is increased by the presence of more than one specie in the system (microcosm). With respect to Zn levels, two of the organisms tend to lose this metal in isolated and microcosm models, probably as a regulation strategy in its accumulation, as well as Fe presence in the reservoir sediments. Therefore...

En los cuerpos acuáticos la disponibilidad de los tóxicos es limitada por las características fisicoquímicas de los sedimentos y agua, así como por las interacciones entre los diversos xenobióticos y entre las especies que los habitan. Por lo anterior, el objetivo del presente trabajo fue relacionar el efecto producido por el zinc (Zn) sembrado en sedimentos del embalse Ignacio Ramírez (PIR), en modelos aislados y en microcosmos, sobre el ATP de res organismos bentónicos con la biodisponibilidad del metal. Las especies seleccionadas fueron: Hyalella azteca, Limnodrilus hoffmeisteri y Stagnicola attenuata, dado que se encuentran en altas proporciones en el embalse y ocupan espacios diferentes en el bentos. Las muestras de sedimentos y organismos se colectaron en la estación de sequía (febrero de 1999). A los primeros se les determinó el pH, textura, tamaño de partícula, nitrógeno total, materia orgánica y concentración de metales (Zn, Fe, Cu y Ni). Posteriormente se realizó un estudio de toxicidad subletal utilizando dos tipos de sistemas estáticos (organismos aislados y en microcosmos). Ambos modelos contenían sedimentos de la PIR enriquecidos con Zn (concentración nominal de 0.8129 mg/kg) y agua sintética en una proporción de 1:4. Los organismos de prueba fueron adicionados una vez alcanzado el equilibrio (2 hr) considerando la cantidad de biomasa con respecto al volumen (1.0 g de organismo por cada 100 ml de agua:sedimento). Después de 0, 12, 24, 36, 48 y 72 hr de exposición se tomaron muestras del sedimento y de los hidrobiontes, a los cuales se les cuantificó el contenido de Zn por absorción atómica. A los organismos también se les determinó la concentración de ATP. Los resultados obtenidos muestran que el efecto producido por los sedimentos naturales enriquecidos se ve potenciado por la presencia de más de una especie en el sistema (microcosmos). Con respecto a los niveles de Zn, dosde los organismos (L. hoffmeisteri y S. attenuata) tienden a...

Effect of vermicomposting process on metal content of paper mill solid waste.

In the present study the change in metal content on vermicomposting of paper mill solid waste was studied. On vermicomposting, quantity of iron, zinc and chromium found to be increased whereas quantity of aluminium, copper, manganese, nickel and lead found comparatively low. The bioaccumulation of any metal was not observed in the body of the earthworm, hence after vermicomposting, there is no harm to fishes to utilize earthworm biomass as their food. The metal content in the vermicompost prepared from the paper mill solid waste was well within the permissive limits and can therefore be safely applied in an aquaculture, agriculture and other food production systems.

Reconfirmation of antimicrobial activity in the coelomic fluid of the earthworm Eisenia fetida andrei by colorimetric assay.

A novel tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) was used in the assessment of antimicrobial activity in earthworm in the presence of phenazine methosulphate (PMS) as an electron coupling reagent. This activity was purified from the coelomic fluid of the earthworm (ECF), Eisenia fetida andrei (Oligochaeta, Lumbricidae, annelids) using a series of column chromatography techniques and was tested against three Gram-negative strains of Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and three Gram-positive strains of Staphylococcus aureus, Bacillus megaterium, Arthrobacter sp., respectively. Only the pigment-free eluate of coelomic fluid of the earthworm (ECFPE) showed activity against B. megaterium amongst three isolated active fractions. The anion (DEAE-52) exchange effluent of the ECFPE was reported to have the strongest activity against P. aeruginosa amongst the three active fractions. The 20% acetonitrile eluate (AE) by Sep-Pak C18 cartridge was also tested and showed fair resistance against E. coli, P. aeruginosa and Arthrobacter sp., respectively.

Mass distributions of a macromolecular assembly based on electrospray ionization mass spectrometric masses of the constituent subunits.

Macromolecular assemblies containing multiple protein subunits and having masses in the megadalton (MDa) range are involved in most of the functions of a living cell. Because of variation in the number and masses of subunits, macromolecular assemblies do not have a unique mass, but rather a mass distribution. The giant extracellular erythrocruorins (Ers), approximately 3.5 MDa, comprised of at least 180 polypeptide chains, are one of the best characterized assemblies. Three-dimensional reconstructions from cryoelectron microscopic images show them to be hexagonal bilayer complexes of 12 subassemblies, each comprised of 12 globin chains, anchored to a subassembly of 36 nonglobin linker chains. We have calculated the most probable mass distributions for Lumbricus and Riftia assemblies and their globin and linker subassemblies, based on the Lumbricus Er stoichiometry and using accurate subunit masses obtained by electrospray ionization mass spectrometry. The expected masses of Lumbricus and Riftia Ers are 3.517 MDa and 3.284 MDa, respectively, with a possible variation of approximately 9% due to the breadth of the mass distributions. The Lumbricus Er mass is in astonishingly good agreement with the mean of 23 known masses, 3.524 +/- 0.481 MDa.

Obtención, composición química, funcional, perfiles electroforéticos y calidad bacteriológica de la carne y harina de lombriz "Eisenia faetida" / Obtainment, chemical composition, functional, electrophoretic profiles and bacteriological quality of the meet and earthworm flour \"Eisenia faetida\"

La harina de Eisenia faetida obtenida a partir de lombrices alimentadas con material orgánico (estiércol de bovino) ha sido analizada para determinar su contenido en nutrientes, encontrándose valores importantes en el porcentaje de proteinas (65 por ciento), grasas (10 por ciento) y minerales (8.8 por ciento). El valor calórico de la harina de lombriz (4 Kca/gr) es similar a la de leche en polvo (4.9 Kcal/gr). El estudio bacteriológico realizado a la harina nos permite afirmar que el producto obtenido presenta un riesgo mínimo y, que ésta materia prima puede ser utilizada en la formulación de diferentes productos destinados a la alimentación humana. El potencial uso de la harina de lombriz es sustentado por la necesidad de enriquecer diferentes formulaciones alimenticias con proteínas no convencionales