Fluorescence-based proteasome activity profiling

With the proteasome emerging as a therapeutic target for cancer treatment, accurate tools for monitoring proteasome (inhibitor) activity are in demand. In this chapter, we describe the synthesis and use of a fluorescent proteasome activity probe that allows for accurate profiling of proteasomal activity in cell lysates, intact cells, and murine and human patient-derived material, with high sensitivity using SDS-PAGE. The probe allows for direct scanning of the gel for fluorescent emission of the distinct proteasomal subunits and circumvents the use of Western blot analysis. Due to its suitable biochemical and biophysical properties, the fluorescent probe can also be used for confocal laser scanning microscopy and flow cytometry-based experiments

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History and International Relation

The first cytoplasmic loop of the mannitol permease from Escherichia coli is accessible for sulfhydryl reagents from the periplasmic side of the membrane

The mannitol permease (EIIMtl) from Escherichia coli couples mannitol transport to phosphorylation of the substrate. Renewed topology prediction of the membrane-embedded C domain suggested that EIIMtl contains more membrane-embedded segments than the six proposed previously on the basis of a PhoA fusion study. Cysteine accessibility was used to confirm this notion. Since cysteine 384 in the cytoplasmic B domain is crucial for the phosphorylation activity of EIIMtl, all cysteine mutants contained this activity-linked cysteine residue in addition to those introduced for probing the membrane topology of the protein. To distinguish between the activity-linked cysteine and the probed cysteine, either trypsin was used to specifically digest the two cytoplasmic domains (A and B), thereby removing Cys384, or Cys384 was protected by phosphorylation from alkylation by N-ethylmaleimide (NEM). Our data show that upon phosphorylation EIIMtl undergoes major conformational changes, whereby residues in the putative first cytoplasmic loop become accessible to NEM. Other residues in this loop were accessible to NEM in intact cells and inside-out membrane vesicles, but cysteine residues at these positions only reacted with the membrane-impermeable sulfhydryl reagent from the periplasmic side of the protein. These and other results suggest that the predicted loop between TM2 and TM3 may fold back into the membrane and form part of the translocation path

How young adults view older people: exploring the pathways of constructing a group image after participation in an intergenerational programme

In recent years, a multitude of intergenerational contact programmes and interventions has emerged to counteract ageism among young adults. Research on these programmes and its supposed effect on ageism often start from the assumption that intergenerational contact follows a largely linear process in which a high level of encounters, in the right setting, decreases ageism and negative stereotyping. The purpose of this article is to critically examine this assumption by focussing on the underlying process of intergenerational contact, rather than examining the positive or negative outcome. Using in-depth interviews with 35 young adults, we found that although conditions and mediating factors during the contact do play a role in the outcome of intergroup contact, the process of contact is rather varied and does not follow a linear path. The results reveal that whether or not a positive contact experience translates into a changed group image of older people is related to the positioning of such experience within the young adults? personal frame of reference. We found that this has to do with the young adults having diverse and both positive and negative previous experiences, their grandparent-grandchild relationship, stories from others and personal characteristics. With this study, we point to the complexity of intergenerational contact and highlight potential pathways leading to varying group images of ?the old?.Prevention, Population and Disease management (PrePoD)Public Health and primary car