Development of a multiple-bile-ion-sensing membrane electrode.

A multiple-bile-ion-sensing polyvinyl chloride-based membrane electrode capable of monitoring any of the three common bile ions in humans, namely, cholate, deoxycholate, and chenodeoxycholate, was developed and characterized. Compared to single-bile-ion-sensing electrodes, it showed a sub-Nernstian response. All other electrode properties were, however, similar, making this a successful replacement for three individual electrodes. With appropriate conditioning, this electrode could repeatedly change selectivity without losing membrane activity. It was reproducible, was stable for 5 months, had low response time, and could be used to measure critical micelle concentrations. The lower limit of detection was 10 nM. Selectivity coefficients for various anions with respect to bile ions more or less followed the Hoffmeister series. Plots of R ((Nernst equivalent of slope in the presence of primary ion and a fixed amount of interfering ion)/(slope in the presence of only the primary ion)) vs square root of ionic strength for an interfering ion were linear. One major application of this electrode is its use in kinetics. We have tested its ability to monitor continuously changing bile ion concentrations during their interactions with a biocompatible polymer, polyethylene glycol (6000), and determined rate constants.

An improved short-lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae.

Ideal reporter genes for temporal transcription programmes have short half-lives that restrict their detection to the window in which their transcripts are present and translated. In an effort to meet this criterion for reporters of transcription in individual living cells, we adapted the ubiquitin fusion strategy for programmable N-end rule degradation to generate an N-degron version of green fluorescent protein (GFP) with a half-life of ~7 min. The GFP variant we used here (designated GFP*) has excellent fluorescence brightness and maturation properties, which make the destabilized reporter well suited for tracking the induction and attenuation kinetics of gene expression in living cells. These attributes are illustrated by its ability to track galactose- and pheromone-induced transcription in S. cerevisiae. We further show that the fluorescence measurements using the short-lived N-degron GFP* reporter gene accurately predict the transient mRNA profile of the prototypical pheromone-induced FUS1 gene.

Optimization of filaggrin expression and processing in cultured rat keratinocytes.

BACKGROUND: In normal mammalian epidermis, cell division occurs primarily in the basal layer where cells are attached to the basement membrane. Upon release from the basement membrane, these basal cells stop dividing and begin to differentiate and stratify producing cornified cells expressing differentiation markers, including the keratin bundling protein filaggrin, and cornified envelope proteins. Little is understood about the regulatory mechanisms of these processes. A rat epidermal keratinocyte cell line synthesizing and processing profilaggrin at confluence in a synchronous manner for 4-5 days provides a useful culture model for epidermal differentiation. Profilaggrin expression in this cell line however decreases with passaging, and its processing involves extensive nonspecific proteolysis. OBJECTIVE: Our objective was to identify culture conditions that effect the decrease in profilaggrin expression with passaging and nonspecific proteolysis of profilaggrin in order to study epidermal differentiation more closely. METHOD: The large amount of nonspecific proteolysis suggested autophagocytosis. To test this, cells were cultured in the presence of 3-methyladenine (3-MA). Two known gradients in epidermis are decreasing serum components and increasing calcium concentrations in the upper cell layers. To determine whether these gradients effected processing, cells were cultured in serum/DMEM or in serum-free KGM and under varying external calcium concentrations. Cells were also cultured in presence of aminoguanidine in an attempt to maintain profilaggrin expression with passaging. RESULTS: Profilaggrin expression was enhanced in the presence of 3-MA, with optimum around 6mM. In the absence of aminoguanidine, profilaggrin expression decreased as a function of increasing passage number; in its presence, profilaggrin expression remained high in some, but not in all of the independently maintained cell lines. Thus, culturing in aminoguanidine was necessary, but not sufficient, for sustained ability to express profilaggrin at confluence. Production of filaggrin from profilaggrin was maximized in a serum-free medium with [Ca(2+)] at 5mM. Filaggrin associates with phospholipid vesicles in vitro forming aggregates similar to those seen in vivo, suggesting that filaggrin release induces vesicular aggregation and autophagocytosis. CONCLUSION: We have used a keratinocyte cell line that synthesizes and processes profilaggrin after confluence as a culture model to study epidermal differentiation. In this system profilaggrin processing must be preceded by inhibition of autophagosome formation and/or modulation of vesicular trafficking, and these processes are regulated by epidermal calcium and serum factor gradients.