Mass spectrometric analysis of electrophoretically separated allergens and proteases in grass pollen diffusates

BACKGROUND: Pollens are important triggers for allergic asthma and seasonal rhinitis, and proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of mucosal epithelial integrity by proteases released by inhaled pollens could promote allergic sensitisation. METHODS: Pollen diffusates from Kentucky blue grass (Poa pratensis), rye grass (Lolium perenne) and Bermuda grass (Cynodon dactylon) were assessed for peptidase activity using a fluorogenic substrate, as well as by gelatin zymography. Following one- or two-dimensional gel electrophoresis, Coomassie-stained individual bands/spots were excised, subjected to tryptic digestion and analysed by mass spectrometry, either MALDI reflectron TOF or microcapillary liquid chromatography MS-MS. Database searches were used to identify allergens and other plant proteins in pollen diffusates. RESULTS: All pollen diffusates tested exhibited peptidase activity. Gelatin zymography revealed high M(r )proteolytic activity at ~ 95,000 in all diffusates and additional proteolytic bands in rye and Bermuda grass diffusates, which appeared to be serine proteases on the basis of inhibition studies. A proteolytic band at M(r )~ 35,000 in Bermuda grass diffusate, which corresponded to an intense band detected by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen Phl p 1, was identified by mass spectrometric analysis as the group 1 allergen Cyn d 1. Two-dimensional analysis similarly demonstrated proteolytic activity corresponding to protein spots identified as Cyn d 1. CONCLUSION: One- and two-dimensional electrophoretic separation, combined with analysis by mass spectrometry, is useful for rapid determination of the identities of pollen proteins. A component of the proteolytic activity in Bermuda grass diffusate is likely to be related to the allergen Cyn d 1

A competência técnica em informática de alunos de enfermagem

Os enfermeiros na era da informação precisam desenvolver seus conhecimentos e habilidades para que se tornem competentes nessa área. O objetivo deste estudo foi verificar o conhecimento dos alunos matriculados no primeiro e no oitavo semestres do curso de graduação em enfermagem dos anos de 2008 e 2007, respectivamente, no que se refere à utilização de recursos da informática. Trata-se de uma pesquisa não experimental do tipo estudo survey descritivo exploratório usado para a coleta dos dados em um questionário baseado em um conjunto de competências em informática. Os resultados mostraram o baixo índice de conhecimentos em informática dos alunos que estão ingressando no curso de graduação. Contudo, na comparação dos aplicativos que os alunos têm maior dificuldade, entre os dois períodos avaliados, a maior porcentagem foi de alunos do oitavo semestre, demonstrando a necessidade da introdução do uso do computador na formação desses novos profissionais para sua posterior adaptação ao mercado de trabalho

High Fat Diet Exacerbates Neuroinflammation in an Animal Model of Multiple Sclerosis by Activation of the Renin Angiotensin System

Epidemiological studies suggest a positive correlation between the incidence and severity of multiple sclerosis (MS) and the intake of fatty acids. It remains to be clarified whether high fat diet (HFD) indeed can exacerbate the disease pathology associated with MS and what the underlying mechanisms are. In this study, we determined the influence of HFD on the severity and pathology of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Mice were fed either normal diet (ND) or HFD and subsequently induced with EAE. Immunohistochemical staining and real-time PCR were used to determine immune cell infiltration and inflammatory mediators in the central nervous system (CNS). Our data show that HFD increases immune cell infiltration and inflammatory mediator production in the CNS and thereby aggravates EAE. Moreover, our data demonstrate that activation of the renin angiotensin system (RAS) is associated with the HFD-mediated effects on EAE severity. These results show that HFD exacerbates an autoreactive immune response within the CNS. This indicates that diets containing excess fat have a significant influence on neuroinflammation in EAE, which may have important implications for the treatment and prevention of neuroinflammatory disorders.We thank W. Leyssens, K. Wauterickx and A. Kerksiek for their technical assistance and animal handling. This work was supported by grants from the Agentschap voor Innovatie door Wetenschap en Technologie (IWT) and Fonds Wetenschappelijk Onderzoek (FWO).experimental autoimmune encephalomyelitis; renin angiotensin system; inflammation; high fat die