Recent studies demonstrate roles for activation of caspases and cleavage of cellular proteins within neurons of the Alzheimer's disease (AD) brain. To determine whether a similar role for caspases also occurs within glial cells in AD, we designed a site-directed caspase-cleavage antibody specific to glial fibrillary acidic protein (GFAP), a cytoskeleton protein specifically expressed in astrocytes. In vitro characterization of this antibody using both a cell-free system and a cell model system of apoptosis demonstrated that the antibody (termed GFAP caspase-cleavage product antibody or GFAP-CCP Ab) immunolabeled the predicted caspase-cleavage fragment, but not full-length GFAP, by Western blot analysis. To determine whether caspases cleave GFAP in vivo, tissue sections from control and AD brains were examined by immunohistochemistry using the GFAP-CCP Ab. Two prominent features of staining were evident: immunolabeling of degenerating astrocytes in proximity to blood vessels and staining within plaque-rich regions of the AD brain. Furthermore, co-localization of the GFAP-CCP Ab and an antibody specific to active caspase-3 was demonstrated within damaged astrocytes of the AD brain. These data suggest that the activation of caspases and cleavage of cellular proteins such as GFAP may contribute to astrocyte injury and damage in the AD brain.
SynopsisObjectiveIn humans, the process of hair shedding, referred to as exogen, is believed to occur independently of the other hair cycle phases. Although the actual mechanisms involved in hair shedding are not fully known, it has been hypothesized that the processes leading to the final step of hair shedding may be driven by proteases and/or protease inhibitor activity. In this study, we investigated the presence of proteases and protease activity in naturally shed human hairs and assessed enzyme inhibition activity of test materials.MethodsWe measured enzyme activity using a fluorescence-based assay and protein localization by indirect immunohistochemistry (IHC). We also developed an ex vivo skin model for measuring the force required to pull hair fibres from skin.ResultsOur data demonstrate the presence of protease activity in the tissue material surrounding club roots. We also demonstrated the localization of specific serine protease protein expression in human hair follicle by IHC. These data provide evidence demonstrating the presence of proteases around the hair club roots, which may play a role during exogen. We further tested the hypothesis that a novel protease inhibitor system (combination of Trichogen® and climbazole) could inhibit protease activity in hair fibre club root extracts collected from a range of ethnic groups (UK, Brazil, China, first-generation Mexicans in the USA, Thailand and Turkey) in both males and females. Furthermore, we demonstrated that this combination is capable of increasing the force required to remove hair in an ex vivo skin model system.ConclusionThese studies indicate the presence of proteolytic activity in the tissue surrounding the human hair club root and show that it is possible to inhibit this activity with a combination of Trichogen® and climbazole. This technology may have potential to reduce excessive hair shedding.RésuméObjectifChez l'homme, le processus de perte de cheveux, désigné comme exogène, est censé se produire indépendamment des autres phases du cycle de cheveux. Bien que les mécanismes réels impliqués dans la perte de cheveux ne soient pas entièrement connus, il a été émis l'hypothèse que les processus conduisant à l'étape finale de la perte de cheveux peuvent être modulés par des protéases et/ou l'activité d'inhibiteurs de protéase. Dans cette étude, nous avons étudié la présence de protéases et de l'activité des protéases dans les cheveux humains perdus naturellement et évalué l'activité inhibitrice d'enzyme de différents matériaux.MéthodesNous avons mesuré l'activité enzymatique en utilisant un dosage basé sur la fluorescence et la localisation des protéines par immunohistochimie indirecte (IHC). Nous avons également développé un modèle de peau ex vivo pour mesurer la force nécessaire pour extraire les fibres capillaires de la peau.RésultatsNos données démontrent la présence d'une activité de la protéase dans le matériau de tissu entourant les racines du bulbe. Nous avons également démontré la localisation de l'expression des protéines de la sérine ...
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