Most eukaryotic cells contain varying amounts of cytosolic lipidic inclusions termed lipid bodies (LBs) or lipid droplets (LDs). In mammalian cells, such as macrophages, these lipid-rich organelles are formed in response to host-pathogen interaction during infectious diseases and are sites for biosynthesis of arachidonic acid (AA)-derived inflammatory mediators (eicosanoids). Less clear are the functions of LBs in pathogenic lower eukaryotes. In this study, we demonstrated that LBs, visualized by light microscopy with different probes and transmission electron microscopy (TEM), are produced in trypomastigote forms of the parasite Trypanosoma cruzi, the causal agent of Chagas’ disease, after both host interaction and exogenous AA stimulation. Quantitative TEM revealed that LBs from amastigotes, the intracellular forms of the parasite, growing in vivo have increased size and electron-density compared to LBs from amastigotes living in vitro. AA-stimulated trypomastigotes released high amounts of prostaglandin E2 (PGE2) and showed PGE2 synthase expression. Raman spectroscopy demonstrated increased unsaturated lipid content and AA incorporation in stimulated parasites. Moreover, both Raman and MALDI mass spectroscopy revealed increased AA content in LBs purified from AA-stimulated parasites compared to LBs from unstimulated group. By using a specific technique for eicosanoid detection, we immunolocalized PGE2 within LBs from AA-stimulated trypomastigotes. Altogether, our findings demonstrate that LBs from the parasite Trypanosoma cruzi are not just lipid storage inclusions but dynamic organelles, able to respond to host interaction and inflammatory events and involved in the AA metabolism. Acting as sources of PGE2, a potent immunomodulatory lipid mediator that inhibits many aspects of innate and adaptive immunity, newly-formed parasite LBs may be implicated with the pathogen survival in its host.
Schistosomiasis is a neglected tropical disease of a significant public health impact. The water rat Nectomys squamipes is one of the most important non-human hosts in the schistosomiasis mansoni transmission in Brazil, being considered a wild reservoir. Cellular mechanisms that contribute to the physiological adaptation of this rodent to the Schistosoma mansoni parasite are poorly understood. Here we identified, for the first time, that a hepatic steatosis, a condition characterized by excessive lipid accumulation with formation of lipid droplets (LDs) within hepatocytes, occurs in response to the natural S. mansoni infection of N. squamipes, captured in an endemic region. Significant increases of LD area in the hepatic tissue and LD numbers/hepatocyte, detected by quantitative histopathological and ultrastructural analyses, were paralleled by increased serum profile (total cholesterol and triglycerides) in infected compared to uninfected animals. Raman spectroscopy showed high content of polyunsaturated fatty acids (PUFAs) in the liver of both groups. MALDI-TOFF mass spectroscopy revealed an amplified pool of omega-6 PUFA arachidonic acid in the liver of infected animals. Assessment of liver functional activity by the levels of hepatic transaminases (ALT and AST) did not detect any alteration during the natural infection. In summary, this work demonstrates that the natural infection of the wild reservoir N. squamipes with S. mansoni elicits hepatic steatosis in the absence of liver functional harm and that accumulation of lipids, markedly PUFAs, coexists with low occurrence of inflammatory granulomatous processes, suggesting that lipid stores may be acting as a protective mechanism for dealing with the infection.
Angular intensity distribution of molecular ions ejected in matrix-assisted laser desorption/ionization (MALDI) have been measured for two symmetrical positions of the laser beam with respect to the sample surface normal (+60" and -60"). It is shown that the molecular ions are ejected in the direction opposite to the incident laser beam with an average angle of -30" which does not depend on the molecular weight in the mass range mlz = 1000 to 25 000. Experiments were performed with the matrix a-cyano4-hydroxy-cinnamic acid.Matrix-assisted laser desorption/ionization (MALDI)',2.3 is now widely used in mass spectrometry of high mass molecules. The ion emission mechanism is related to the initial physical parameters at the time of ejection and several authors have recently measured the axial and radial ion v e l o c i t i e~.~.~ Angular distributions of emitted ions were also measured in MALDI with dihydroxybenzoic acid (DHB) as a matrix and at a fixed angle between the sample surface and the laser beam.6 In Ref. 7 it is shown that the ion yield in MALDI, measured as a function of the laser beam incident angle and laser energy, does not depend on the angle. In all these experiments the'laser beam axis was kept on one side of the sample with respect to the surface normal. The present work shows results on angular intensity distribution of ions in MALDI, obtained at laser beam angles of +60" and -60". The sample was at a fixed position (perpendicular to the time-of-flight (TOF) mass spectrometer axis) and the laser could easily be changed from one side to the other side of the TOF axis, all other experimental conditions remaining the same. EXPERIMENTALThe experimental arrangement is shown in Fig. 1. A 252Cf source and a nitrogen UV laser can be used as desorption probes in a linear time-of-flight spectrometer. The 252Cf source is centered 5 m m behind the sample. At the extremity of the TOF tube, a dual microchannel plate (MCP) detector can move, along an axis (X-axis) perpendicularly to the TOF axis in the plane formed by the TOF axis (Z-axis) and the X-axis. This plane (X-Z) contains the laser beam axis. It can also move in a direction Y, perpendicular to this plane. A collimator of 5 mm diameter was attached to the detector and the assembly was moved by steps of 5 mm. The secondary-ion intensity can thus be measured as a function of the distance dx to the TOF axis. The distance from the detector to the sample was 75 cm. A grid extractor, at ground potential, was at 9 mm from the sample, where an extraction voltage between 7 and 15 kV was applied. In order to define precisely the Author for correspondence. time-of-flight axis, the secondary-ion intensity distributions of cesium iodide clusters produced in the bombardment of a CsI layer by fission fragments from 252Cf was measured. Cs+ ions, as well as cluster ions (CsI),Cs (with n = 1 to 3) have the same intensity distribution, as shown in Fig. 2(a) ((X-Z) plane). A simple correction was applied to the measured values to take into account the collimator diameter and...
With the aim of studying the interaction of fast electrons with solid surfaces we have developed an experimental set-up based on electron stimulated desorption (ESD) coupled to time-of-flight (TOF) mass spectrometry. Poly(methyl methacrylate) and poly(vynil chloride) samples have been irradiated by a pulsed electron beam of 1.2 keV and 0.18 µs FWHM. The results show that H + is the main ionic species to desorb after electron bombardment. In addition, other ionic fragments were also observed and assigned. These results show the potentiality of this technique in the study of ESD of polymers.Keywords: electron stimulated desorption; time-of-flight spectrometer; polymers. INTRODUÇÃOPara os pesquisadores que atuam na área de Ciência de Superfí-cies é muito importante obter um conhecimento elementar da superfície em estudo e mais importante ainda é o conhecimento das espé-cies moleculares, que estão na superfície. Uma das técnicas mais poderosas de análise superficial é a Espectroscopia Auger (AES -"Auger Electron Spectroscopy") 1 . Esta técnica fornece informação elementar, com exceção do hidrogênio e do hélio. Todavia, ela não fornece informação em relação ao estado químico das espécies adsorvidas na superfície, ou seja, espécies moleculares na superfície não podem ser detectadas. Outra técnica que fornece informação química direta sobre a superfície é a dessorção induzida por laser utilizando uma matriz especial, que absorve a radiação laser (MALDI -"Matrix-Assisted Laser Desorption Ionization") 2-4 . Apesar de ser possível atualmente fazer uma varredura do laser pela superfície com resoluções laterais da ordem de mícrons 4 , a técnica necessita de uma matriz, o que elimina a possibilidade de se estudar amostras in situ, além de ser altamente destrutiva. Uma das técnicas que mais se desenvolveu nos últimos 20 anos na área de análise de superfícies é a técnica de espectrometria de massas de íons secundários formados a partir do impacto de íons primários monoatômicos ou poliatômicos com a superfície (SSIMS -"Static Secondary Ion Mass Spectrometry") 5-7 . Esta técnica pode fornecer informação química de monocamadas adsorvidas sobre todos os tipos de superfícies e pode vir a ser a técnica preferida para análise química superficial em um futuro próximo. Entretanto, esta técnica também apresenta desvantagens: o baixo rendimento dos íons secundários, o efeito da matriz e as grandes seções de choque de dano, que limitam o uso da técnica abaixo do limite estático 7 , onde uma corrente de íons primários muito baixa tem que ser usada (nunca maior que 10 13 íons.cm -2 ), com a finalidade de garantir que menos de 1% dos átomos ou moléculas superficiais recebam um impacto iônico.A utilização de elétrons rápidos apresenta uma série de vantagens com respeito às técnicas analíticas mencionadas acima. Em primeiro lugar, o dano produzido por elétrons nas amostras é muito menor que aquele decorrente de um laser ou de um íon de alta energia [8][9] . Segundo, é de se esperar que o efeito de matriz seja menor que no caso de SSIMS, devido...
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