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1.
Appl Radiat Isot ; 148: 204-212, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30981125

RESUMO

A cylindrical graphite illuminator with a thickness of 6.5 cm and diameter of 18 cm was installed inside the collimator of INUS (Instalatie de Neutronografie UScata) neutron imaging facility in the past. The graphite illuminator is usually utilized inside the collimator of neutron imaging facility to provide an intense and approximately uniform beam of neutrons at the outlet of collimator. With the mentioned existing illuminator in INUS imaging facility, the thermal neutron flux at the exit of collimator was measured 7.2 × 104 n/cm2/s. Also the obtained neutron beam profile in this facility shows that it is not completely uniform at the imaging screen and the intensity of neutrons at the top and bottom of beam profile are not the same. Hence, in this paper a new graphite illuminator is proposed to improve the neutron beam characteristics in INUS imaging facility. Monte Carlo N-Particle (MCNP) code was implemented in this study for evaluating the proposed illuminator. The shape of proposed illuminator is a cylinder whose one side is inclined. Three quality factors of thermal neutron intensity, thermal neutron beam uniformity and gamma radiation dose rate were used to evaluate performance of the new illuminator. In order to obtain optimum illuminator shape, three effective parameters of thickness, angle of inclined side and position of the illuminator inside the collimator were investigated in this research. The investigation was carried out on thicknesses in the range of 5 to 25 cm with a step of 5 cm, angles in the range of 10 to 60° with a step of 10° and positions of -5, 0 and 5 cm with respect to center of reactor core. After investigating and interpolating the results, it was found that the proposed illuminator with a thickness of 10 cm, angle of 54.5° and position of 0 can produce a uniform beam profile, increase the thermal neutron intensity up to 7.1% and also decrease the neutron to gamma ratio up to 5% in comparison with the existing one.

2.
Appl Radiat Isot ; 139: 294-303, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29883948

RESUMO

Simulating X-ray images is of great importance in industry and medicine. Using such simulation permits us to optimize parameters which affect image's quality without the limitations of an experimental procedure. This study revolves around a novel methodology to simulate a complete industrial X-ray digital radiographic system composed of an X-ray tube and a computed radiography (CR) image plate using Monte Carlo N Particle eXtended (MCNPX) code. In the process of our research, an industrial X-ray tube with maximum voltage of 300 kV and current of 5 mA was simulated. A 3-layer uniform plate including a polymer overcoat layer, a phosphor layer and a polycarbonate backing layer was also defined and simulated as the CR imaging plate. To model the image formation in the image plate, at first the absorbed dose was calculated in each pixel inside the phosphor layer of CR imaging plate using the mesh tally in MCNPX code and then was converted to gray value using a mathematical relationship determined in a separate procedure. To validate the simulation results, an experimental setup was designed and the images of two step wedges created out of aluminum and steel were captured by the experiments and compared with the simulations. The results show that the simulated images are in good agreement with the experimental ones demonstrating the ability of the proposed methodology for simulating an industrial X-ray imaging system.

3.
Int J Phytoremediation ; 18(4): 337-47, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26540563

RESUMO

Phytoremediation potential of the aquatic plant Spirodela polyrrhiza was examined for direct blue 129 (DB129) azo dye. The dye removal efficiency was optimized under the variable conditions of the operational parameters including removal time, initial dye concentration, pH, temperature and amount of plant. The study reflected the significantly enhanced dye removal efficiency of S. polyrrhiza by increasing the temperature, initial dye concentration and amount of plant. Intriguingly, artificial neural network (ANN) predicted the removal time as the most dominant parameter on DB129 removal efficiency. Furthermore, the effect of dye treatment on some physiologic indices of S. polyrrhiza including growth rate, photosynthetic pigments content, lipid peroxidation and antioxidant enzymes were studied. The results revealed a reduction in photosynthetic pigments content and in multiplication of fronds after exposure to dye solution. In contrast, malondialdehyde content as well as catalase (CAT) and peroxidase (POD) activities significantly increased that was probably due to the ability of plant to overcome oxidative stress. As a result of DB129 biodegradation, a number of intermediate compounds were identified by gas chromatography-mass spectroscopy (GC-MS) analysis. Accordingly, the probable degradation pathway of DB129 in S. polyrrhiza was postulated.


Assuntos
Araceae/metabolismo , Corantes/metabolismo , Recuperação e Remediação Ambiental/métodos , Araceae/química , Araceae/enzimologia , Araceae/genética , Biodegradação Ambiental , Clorofila/metabolismo , Corantes/química , Malondialdeído/metabolismo , Redes Neurais de Computação , Peroxidases/genética , Peroxidases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
4.
Int J Phytoremediation ; 17(1-6): 322-9, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25409244

RESUMO

The present study was conducted to evaluate the potential of aquatic vascular plant, Nasturtium officinale, for degradation of C.I. Acid Blue 92 (AB92). The effect of operational parameters such as initial dye concentration, plant biomass, pH, and temperature on the efficiency of biological decolorization process was determined. The reusability of the plant in long term repetitive operations confirmed the biological degradation process. The by-products formed during biodegradation process were identified by GC-MS technique. The effects of the dye on several plant physiological responses such as photosynthetic pigments content and antioxidant enzymes activity were investigated. The content of chlorophyll and carotenoids was significantly reduced at 20 mg/L of the dye. The activities of superoxide dismutase and peroxidase were remarkably increased in the plant root verifying their importance in plant tolerance to the dye contamination.


Assuntos
Corantes/metabolismo , Naftalenos/metabolismo , Nasturtium/metabolismo , Biodegradação Ambiental , Carotenoides/metabolismo , Catalase/metabolismo , Clorofila/metabolismo , Corantes/química , Estrutura Molecular , Naftalenos/química , Nasturtium/enzimologia , Peroxidases/metabolismo , Proteínas de Plantas/metabolismo , Superóxido Dismutase/metabolismo
5.
Int J Phytoremediation ; 15(8): 729-42, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23819271

RESUMO

The potential of an aquatic fern, Azolla filiculoides, in phytoremediation of a mono azo dye solution, C.I. Acid Blue 92 (AB92), was studied. The effects of operational parameters such as reaction time, initial dye concentration, fern fresh weight, pH, temperature and reusability of the fern on biodegradation efficiency were investigated. The intermediate compounds produced by biodegradation process were analyzed using GC-MS analysis. An artificial neural network (ANN) model was developed to predict the biodegradation efficiency. The findings indicated that ANN provides reasonable predictive performance (R2 = 0.961). The effects of AB92 solutions (10 and 20 mg L(-1)) on growth, chlorophylls and carotenoids content, activity of antioxidant enzymes such as superoxide dismutase, peroxidase and catalase and formation of malondialdehyde were analyzed. AB92 generally showed inhibitory effects on the growth. Moreover, photosynthetic pigments in the fronds significantly decreased in the treatments. An increase was detected for lipid peroxidation and antioxidant enzymes activity, suggesting that AB92 caused reactive oxygen species production in Azolla fronds, which were scavenged by induced activities of antioxidant enzymes.


Assuntos
Corantes/metabolismo , Gleiquênias/metabolismo , Naftalenos/metabolismo , Redes Neurais de Computação , Antioxidantes/metabolismo , Biodegradação Ambiental , Biomassa , Carotenoides/análise , Carotenoides/metabolismo , Catalase/efeitos dos fármacos , Catalase/metabolismo , Clorofila/análise , Clorofila/metabolismo , Gleiquênias/efeitos dos fármacos , Gleiquênias/crescimento & desenvolvimento , Cromatografia Gasosa-Espectrometria de Massas , Concentração de Íons de Hidrogênio , Peroxidação de Lipídeos/efeitos dos fármacos , Naftalenos/farmacologia , Peroxidases/efeitos dos fármacos , Peroxidases/metabolismo , Proteínas de Plantas/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Temperatura
6.
Ecotoxicol Environ Saf ; 93: 128-34, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23660490

RESUMO

The potential of Hydrocotyle vulgaris as an aquatic plant species was evaluated for phytoremediation of C.I. Basic Red 46 (BR46) from nutrient solution. Under the optimized experimental conditions, BR46 was removed up to 95% from incubation medium by H. vulgaris. The ability of the plant in consecutive removal under long term repetitive experiments confirmed the biodegradation process. Accordingly, a number of produced intermediate compounds were identified. An artificial neural network (ANN) model was developed to predict the biodegradation efficiency. A predictive performance (R(2)=0.974) was obtained based on the network results. Interestingly, dye stress enhanced the activity of antioxidant enzymes including superoxide dismutase, peroxidase and catalase in H. vulgaris roots and leaves. Enzymatic responses found to be highly depended on the plant organ and dye concentration in the liquid medium. Overall, the increase in the activity of antioxidant enzymes was much higher in the roots than in the leaves. Nevertheless, no significant increase in the malondialdehyde (MDA) content was detected in both roots and leaves which reflects the high efficiency of antioxidant system in the elimination of reactive oxygen species.


Assuntos
Centella/fisiologia , Corantes/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Poluentes Químicos da Água/metabolismo , Biodegradação Ambiental , Catalase , Centella/metabolismo , Corantes/análise , Malondialdeído/metabolismo , Modelos Químicos , Redes Neurais de Computação , Peroxidases/metabolismo , Superóxido Dismutase/metabolismo , Têxteis , Eliminação de Resíduos Líquidos/métodos , Águas Residuárias/química , Poluentes Químicos da Água/análise
7.
Ecotoxicol Environ Saf ; 80: 291-8, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22498423

RESUMO

In present study, the potential of duckweed (Lemna minor L.) for degradation of an azo dye C.I. Acid Blue 92 (AB92) has been investigated. The effect of operational parameters such as initial dye concentration, pH, temperature and amount of plant on the efficiency of biological decolorization process was determined. The reusability of Lemna minor L. in long term repetitive operations was also examined. Growth and some biochemical parameters (photosynthetic pigments content, superoxide dismutase, catalase and peroxidase activity) were used to detect the toxic effects of AB92 on duckweed plant. The biological degradation compounds formed in the present process were analyzed by GC-MS technique. In addition, an artificial neural network (ANN) model was expanded to predict the biological decolorization efficiency. The obtained data indicated that ANN provide realistic predictive performance (R(2)=0.954).


Assuntos
Araceae/metabolismo , Naftalenos/metabolismo , Poluentes Químicos da Água/metabolismo , Araceae/crescimento & desenvolvimento , Biodegradação Ambiental , Simulação por Computador , Naftalenos/análise , Redes Neurais de Computação , Temperatura , Indústria Têxtil , Poluentes Químicos da Água/análise
8.
Radiat Prot Dosimetry ; 144(1-4): 239-42, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21199823

RESUMO

In this research work, a semi-automatic densitometry system has been developed for large-scale monitoring services by use of film badge dosemeters. The system consists of a charge-coupled device (CCD)-based scanner that can scan optical densities (ODs) up to 4.2, a computer vision algorithm to improve the quality of digitised films and an analyser program to calculate the necessary information, e.g. the mean OD of region of interest and radiation doses. For calibration of the system, two reference films were used. The Microtek scanner International Color Consortium (ICC) profiler is applied for determining the colour attributes of the scanner accurately and a reference of the density step tablet, Bundesanstalt für Materialforschung und-prüfung (BAM) is used for calibrating the automatic conversion of gray-level values to OD values in the range of 0.2-4.0 OD. The system contributes to achieve more objectives and reliable results. So by applying this system, we can digitise a set of 20 films at once and calculate their relative doses less than about 4 min, and meanwhile it causes to avoid disadvantages of manual process and to enhance the accuracy of dosimetry.


Assuntos
Densitometria/instrumentação , Dosimetria Fotográfica/instrumentação , Monitoramento de Radiação/instrumentação , Proteção Radiológica/instrumentação , Algoritmos , Automação , Calibragem , Computadores , Densitometria/métodos , Desenho de Equipamento , Dosimetria Fotográfica/métodos , Humanos , Processamento de Imagem Assistida por Computador , Teste de Materiais , Doses de Radiação , Monitoramento de Radiação/métodos , Proteção Radiológica/métodos , Reprodutibilidade dos Testes , Software
9.
Nat Prod Res ; 25(16): 1480-7, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20635302

RESUMO

Callus induction and proliferation of Echium italicum L. (Boraginaceae) were investigated using cotyledon, hypocotyl and root explants. Calli were initiated and established using B5, LS, 1/2LS and White media supplemented with different auxins, including 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA) and 1-naphthaleneacetic acid (NAA) in combination with kinetin. The maximum pigmented callus induction (100%) was observed in the White medium. The n-hexane extract of proliferated callus tissues were analysed by TLC and HPLC. The major secondary metabolite was separated by preparative HPLC and its structure was elucidated by UV, ¹H and ¹³C-NMR spectroscopy. As a result, shikonin acetate was identified by various spectroscopic methods from callus culture of E. italicum. These findings highlight the shikonin production potential of the E. italicum callus, which may be considered as a new source for the production of shikonin and its derivatives for industrial use.


Assuntos
Antraquinonas , Echium/química , Técnicas de Cultura de Tecidos/métodos , Antraquinonas/química , Antraquinonas/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Cotilédone/química , Meios de Cultura/química , Hipocótilo/química , Ácidos Indolacéticos/química , Cinetina/química , Espectrometria de Massas , Raízes de Plantas/química , Espectrofotometria Ultravioleta
10.
Nat Prod Res ; 24(14): 1330-6, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20803377

RESUMO

A hydrodistillation sampling method, coupled to gas chromatography-mass spectrometry, was used in monitoring the volatile organic compounds in flowers of Astragalus sahendi. Accordingly, a total of 48 compounds were recognised, which were united by their terpenoid or aliphatic skeletons and low molecular weight. Above all, the significant presence of some insect-favoured terpenoid compounds, such as farnesol, cis- and trans-geraniol, alpha-bisabolol, nerolidol isomer, alpha-terpineol, alpha-terpinolene and thymol was significant. These findings confer a better understanding of pollination processes in the giant genus Astragalus. Furthermore, the results add to an increasing quantity of data corroborating the ecologic and evolutionary correlation between the floral bioactive compounds of plant species and their special types of pollinators.


Assuntos
Astrágalo/química , Flores/química , Terpenos/análise , Compostos Orgânicos Voláteis/análise , Cromatografia Gasosa-Espectrometria de Massas , Irã (Geográfico)
11.
Nat Prod Res ; 24(13): 1235-42, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20645210

RESUMO

The chemical composition of the volatile organic compounds (VOCs) released from the leaves and flowers of Artemisia fragrans Willd. (Asteraceae) was investigated using headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography (GC) and/or GS/mass spectrometry (GC/MS). The efficiency and reliability of different fibres such as modified pencil lead and commercial polyacrylate (PA) as well as polydimethylsiloxane (PDMS) were evaluated, and pencil lead was selected to be the most suitable fibre for the extraction and sampling of the compounds. The extraction and chromatographic conditions were also optimised. Under the optimal conditions, a range of VOCs released from the leaves and flowers of A. fragrans were identified and compared. Accordingly, some bioactive components such as camphor, 1,8-cineole, alpha-terpinolene, gamma-terpinene and carvacrol were the main compounds in the VOCs released from either flowers or leaves. However, the nature and level of the volatiles in studied parts of the plant were notably varied.


Assuntos
Artemisia/química , Flores/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Microextração em Fase Sólida/métodos , Compostos Orgânicos Voláteis/isolamento & purificação , Resinas Acrílicas , Cromatografia Gasosa , Dimetilpolisiloxanos , Cromatografia Gasosa-Espectrometria de Massas , Grafite
12.
Nat Prod Res ; 24(11): 1069-78, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20552530

RESUMO

Two new cycloartane-type glycosides, caspicuside I and caspicuside II, were isolated along with two known saponins, cycloastragenol and astragaloside IV, from the roots of Astragalus caspicus. As the aglycon group, caspicuside I possesses 3beta,6alpha,16beta,(24S),25-pentahydroxycycloartane (cyclocanthagenol), while caspicuside II owns (20R, 24S)-epoxy-3beta, 6alpha,16beta, 25-tetrahydroxycycloartane (cycloastragenol). The chemical structures of these new cycloartane-type glycosides were established as 3-O-alpha-L-rhamnopyranosyl-16-O-beta-D-xylopyranosyl-cyclocanthogenol and 3-O-[beta-D-xylopyranosyl(1 --> 3)-beta-D-glucopyranosyl]-6-O-beta-xylopyranosyl-cycloastragenol, respectively. These findings add to a growing body of literature demonstrating the high variation of cycloartane-type triterpene glycosides in different species of Astragalus.


Assuntos
Astrágalo/química , Glicosídeos/química , Raízes de Plantas/química , Triterpenos/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular
13.
Nat Prod Res ; 24(8): 703-9, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20432151

RESUMO

A solid phase microextraction sampling method using pencil-lead fibre coupled with gas chromatography-mass spectrometry was used to monitor the organic volatile compounds of the roots, leaves and gum of Astragalus compactus. Under optimised conditions, a range of volatile compounds were recognised in different parts of A. compactus. Based on the results obtained, the nature and level of the volatiles vary in distinct parts of the plant. In comparison with other Astragalus species investigated, only one volatile organochlorine compound, tetradecane,1-chloro, was identified in roots. Intriguingly, the toxic chlorinated component was not detectable in the gum samples. These findings highlight the higher quality of the gum of A. compactus compared to the gums of some other Astragalus species, which are reported to be contaminated with chlorinated components.


Assuntos
Astrágalo/química , Folhas de Planta/química , Raízes de Plantas/química , Resinas Vegetais/química , Compostos Orgânicos Voláteis/química , Cromatografia Gasosa-Espectrometria de Massas , Microextração em Fase Sólida
14.
Nat Prod Res ; 23(3): 301-8, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19235031

RESUMO

Chemical composition of the floral nectar of Peganum harmala, a herbaceous medicinal perennial of the family Zygophyllaceae, was analysed using high-performance liquid chromatography technique. The nectar sugar detection experiments resulted in 33.1, 39.8 and 27.4%, respectively, for fructose, glucose and sucrose, upon which the nectar was classified as hexose rich. In addition, 11 proteinaceous amino acids were recognised and quantified in the nectar. Concentration of the insects' favoured amino acid, prolin, was markedly high. Furthermore, among four detected alkaloids, harmalol and harmine as the two beta-carboline derivatives were identified. These findings may confer a better understanding upon outcrossing processes and favour the plant-pollinator relationships.


Assuntos
Flores/química , Peganum/química , Cromatografia Líquida de Alta Pressão , Frutose/química , Glucose/química , Harmalina/análogos & derivados , Harmalina/química , Harmina/química , Sacarose/química
15.
Nat Prod Res ; 22(18): 1660-9, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19085425

RESUMO

A solid phase microextraction sampling method using homemade pencil-lead fibre coupled with gas chromatography was developed as a fast, easy and reliable monitoring technique to recognise the organic volatile compounds of the roots, leaves and gum of Astragalus microcephalus. The efficiency and reliability of different fibres such as homemade pencil-lead and commercial polydimethylsiloxan, as well as polyacrylate, were evaluated and the most effective fibre for recognition of these compounds was selected. Some effective experimental parameters, such as extraction time and chromatographic conditions, were examined and optimised. Under the optimised conditions, various volatile compounds such as 2-hexyn-1-ol, pentanal 3-methyl, caryophyllene, octadecane and some chlorinated components were identified in different parts of A. microcephalus. The results obtained showed that in distinct parts of A. microcephalus, the nature and level of the volatiles were varied.


Assuntos
Astrágalo/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Microextração em Fase Sólida/métodos , Compostos Orgânicos Voláteis/isolamento & purificação , Gomas Vegetais/química , Folhas de Planta/química , Raízes de Plantas/química
16.
Plant Cell ; 13(9): 2005-20, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11549760

RESUMO

COPII-coated vesicles, first identified in yeast and later characterized in mammalian cells, mediate protein export from the endoplasmic reticulum (ER) to the Golgi apparatus within the secretory pathway. In these organisms, the mechanism of vesicle formation is well understood, but the process of soluble cargo sorting has yet to be resolved. In plants, functional complements of the COPII-dependent protein traffic machinery were identified almost a decade ago, but the selectivity of the ER export process has been subject to considerable debate. To study the selectivity of COPII-dependent protein traffic in plants, we have developed an in vivo assay in which COPII vesicle transport is disrupted at two distinct steps in the pathway. First, overexpression of the Sar1p-specific guanosine nucleotide exchange factor Sec12p was shown to result in the titration of the GTPase Sar1p, which is essential for COPII-coated vesicle formation. A second method to disrupt COPII transport at a later step in the pathway was based on coexpression of a dominant negative mutant of Sar1p (H74L), which is thought to interfere with the uncoating and subsequent membrane fusion of the vesicles because of the lack of GTPase activity. A quantitative assay to measure ER export under these conditions was achieved using the natural secretory protein barley alpha-amylase and a modified version carrying an ER retention motif. Most importantly, the manipulation of COPII transport in vivo using either of the two approaches allowed us to demonstrate that export of the ER resident protein calreticulin or the bulk flow marker phosphinothricin acetyl transferase is COPII dependent and occurs at a much higher rate than estimated previously. We also show that the instability of these proteins in post-ER compartments prevents the detection of the true rate of bulk flow using a standard secretion assay. The differences between the data on COPII transport obtained from these in vivo experiments and in vitro experiments conducted previously using yeast components are discussed.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Nicotiana/citologia , Nicotiana/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Ligação ao Cálcio/metabolismo , Calreticulina , Escherichia coli , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina , Ligantes , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Oligopeptídeos/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas/genética , Receptores de Peptídeos/metabolismo , Ribonucleoproteínas/metabolismo , Solubilidade , Especificidade por Substrato , Temperatura , Proteínas de Transporte Vesicular , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/genética , alfa-Amilases/metabolismo
17.
J Cell Biol ; 152(1): 41-50, 2001 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-11149919

RESUMO

Developing pea cotyledons contain functionally different vacuoles, a protein storage vacuole and a lytic vacuole. Lumenal as well as membrane proteins of the protein storage vacuole exit the Golgi apparatus in dense vesicles rather than in clathrin-coated vesicles (CCVs). Although the sorting receptor for vacuolar hydrolases BP-80 is present in CCVs, it is not detectable in dense vesicles. To localize these different vacuolar sorting events in the Golgi, we have compared the distribution of vacuolar storage proteins and of alpha-TIP, a membrane protein of the protein storage vacuole, with the distribution of the vacuolar sorting receptor BP-80 across the Golgi stack. Analysis of immunogold labeling from cryosections and from high pressure frozen samples has revealed a steep gradient in the distribution of the storage proteins within the Golgi stack. Intense labeling for storage proteins was registered for the cis-cisternae, contrasting with very low labeling for these antigens in the trans-cisternae. The distribution of BP-80 was the reverse, showing a peak in the trans-Golgi network with very low labeling of the cis-cisternae. These results indicate a spatial separation of different vacuolar sorting events in the Golgi apparatus of developing pea cotyledons.


Assuntos
Cotilédone/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Transporte Vesicular , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Pisum sativum , Receptores de Superfície Celular/metabolismo , Proteínas de Armazenamento de Sementes , Vacúolos/metabolismo , beta-Frutofuranosidase , Leguminas
18.
Plant Cell ; 12(11): 2219-36, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11090220

RESUMO

Coat protein (COP)-coated vesicles have been shown to mediate protein transport through early steps of the secretory pathway in yeast and mammalian cells. Here, we attempt to elucidate their role in vesicular trafficking of plant cells, using a combined biochemical and ultrastructural approach. Immunogold labeling of cryosections revealed that COPI proteins are localized to microvesicles surrounding or budding from the Golgi apparatus. COPI-coated buds primarily reside on the cis-face of the Golgi stack. In addition, COPI and Arf1p show predominant labeling of the cis-Golgi stack, gradually diminishing toward the trans-Golgi stack. In vitro COPI-coated vesicle induction experiments demonstrated that Arf1p as well as coatomer could be recruited from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/Golgi membranes. Binding of Arf1p and coatomer is inhibited by brefeldin A, underlining the specificity of the recruitment mechanism. In vitro vesicle budding was confirmed by identification of COPI-coated vesicles through immunogold negative staining in a fraction purified from isopycnic sucrose gradient centrifugation. Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley alpha-amylase-HDEL yielded a COPI-coated vesicle fraction that contained alpha-amylase as well as calreticulin.


Assuntos
Proteínas de Plantas/metabolismo , Sequência de Bases , Brefeldina A/farmacologia , Reações Cruzadas , Primers do DNA , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Soros Imunes , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/imunologia , Plantas Geneticamente Modificadas/metabolismo , Transporte Proteico
19.
Plant Physiol ; 119(4): 1437-46, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10198103

RESUMO

Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles. Their existence in plant cells has not yet been demonstrated, although the GTP-binding proteins required for coat formation have been identified. We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins. The amounts of the cytosolic antigens relative to the membrane-bound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins. Sucrose-density-gradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.


Assuntos
Arabidopsis/metabolismo , Proteínas de Plantas/metabolismo , Citosol/metabolismo , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Proteínas de Plantas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Frações Subcelulares/metabolismo
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