1. Conventional and ion-selective double-barrelled microelectrodes were used in an in vitro bovine retinal pigment epithelium (RPE)-choroid preparation to measure the changes in membrane voltage, resistance and intracellular K+ and Cl-activities produced by small, physiological changes in extracellular potassium ([K+]0).2. In the intact eye, light-induced changes in [K+]. occur in the extracellular (or subretinal) space that separates the neural retina and the RPE apical membrane. These [K+1 changes can be approximated in vitro by decreasing apical bath [K+]. from 5 to 2 mM.3. This in vitro change in [K+1 simultaneously decreased intracellular Cl-and K+ activities (a4 and ak) by 25 + 6 mM (n = 8) and 19 + 7 mM (n = 4) (mean + S.D.), respectively.In control Ringer solution (5 mm [K+]0) a', and a' were 65+10 mm (n = 28) and 65 + 8 mM (n = 6), respectively. The [K+]t-induced decreases in a', and a' were both significantly inhibited, either by blocking the apical membrane K+ conductance with Ba2+ or the basolateral membrane Cl-conductance with DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid). 5. Transepithelial current pulses were used to determine the relative basolateral membrane Cl-conductance, T5s, was -0-6 (n = 3), and the relative apical membrane K+ conductance, TK, was -0'7 (n = 2).Step changes in basal bath [Kt]o were used to estimate the relative basolateral membrane K+ conductance, TBAS, was -0 34 (n = 3). 6. These data show that the apical membrane K+ conductance and the basolateral membrane Cl-conductance are electrically coupled. In vivo, this coupling could have significant functional importance by modulating the relative hydration of the subretinal space, regulating RPE cell volume, and subretinal space.In the posterior portion of the vertebrate eye the apical membrane of the retinal pigment epithelium (RPE) and the photoreceptors communicate across a small extracellular or subretinal space. The basolateral membrane faces a different extracellular environment which is produced by the choroidal blood supply. The flow of metabolites, ions and fluid between these extracellular spaces is mediated by a variety of apical and basolateral membrane transport

The transfer-messenger RNA (tmRNA)-mediated trans-translation mechanism is highly conserved in bacteria and functions primarily as a system for the rescue of stalled ribosomes and the removal of aberrantly produced proteins. Here, we show that in the antibiotic-producing soil bacterium Streptomyces coelicolor, trans-translation has a specialized role in stress management. Analysis of proteins that were carboxy-terminally His 8 -tagged by a recombinant tmRNA identified only 10 targets, including the stress proteins: DnaK heat-shock protein 70, thiostrepton-induced protein A, universal stress protein A, elongation factor Tu3, and the cell-cycle control proteins DasR, SsgA, SsgF and SsgR. Although tmRNA-tagged proteins are degraded swiftly, the translation of dnaK and dasR messenger RNAs (mRNAs) depends fully on tmRNA, whereas transcription is unaffected. The data unveil a surprisingly dedicated functionality for tmRNA, promoting the translation of the same mRNA it targets, at the expense of sacrificing the first nascent protein. In streptomycetes, tmRNA has evolved into a dedicated task force that ensures the instantaneous response to the exposure to stress.

Members of the prokaryotic genus Streptomyces produce over 60% of all known antibiotics and a wide range of industrial enzymes. A leading theme in microbiology is which signals are received and transmitted by these organisms to trigger the onset of morphological differentiation and antibiotic production. The small ␥-butyrolactone A-factor is an important autoregulatory signaling molecule in streptomycetes, and A-factor mutants are blocked in development and antibiotic production. In this study we showed that heterologous expression of the 324-amino acid secreted regulatory protein Factor C resulted in restoration of development and enhanced antibiotic production of an A-factor-deficient bald mutant of Streptomyces griseus, although the parental strain lacks an facC gene. Proteome analysis showed that in the facC transformant the production of several secreted proteins that belong to the A-factor regulon was restored. HPLC-MS/MS analysis indicated that this was due to restoration of A-factor production to wild-type levels in the transformant. This indicates a connection between two highly divergent types of signaling molecules and possible interplay between their regulatory networks.

three distinct phases. The first phase was generated by an apical membrane hyperpolarization, followed by a (delayed) basolateral membrane hyperpolarization (DBMH); the third phase was an apical membrane depolarization. The present experiments focus on the membrane and cellular mechanisms that generate phase 2 of the response, the DBMH.3. The DBMH was abolished in the presence of apical bumetanide (100 fM); this response was completely restored after bumetanide removal. Reducing apical [K+]O, adding apical bumetanide (500 mM), or removing apical Cldecreased 41 by 25 + 6 (n = 8), 28 ± 1 (n = 2) and 26 + 5 mM (n = 3), respectively; adding 100 #sm apical bumetanide decreased a{1 by 12 + 2 mM (n = 3). Adding apical bumetanide or removing apical bath CF hyperpolarized the basolateral membrane and decreased the apparent basolateral membrane conductance (GB).

Nonsteroidal anti-inflammatory drugs (NSAIDs) were added to the solutions bathing the apical membrane of bovine retinal pigment epithelium (RPE)-choroid explants. For example, niflumic acid (100 microM) depolarized the basolateral membrane voltage (VB) by approximately 12 mV, increased transepithelial potential by 4.5 mV, decreased intracellular Cl activity by 13 mM, decreased transepithelial resistance by 17 omega.cm2, and increased the ratio of apical to basolateral membrane resistance nearly threefold. All of these changes are consistent with an increase in basolateral membrane Cl conductance. In addition, niflumic acid caused intracellular Ca concentration to decrease by 16 nM and fluid transport rate to increase by 1.5 microliters.cm-2.h-1. Flufenamic acid, which is structurally very similar to niflumic acid, had the opposite effects on membrane voltage and resistance. Basal application of the Cl channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or current clamping VB to the reversal potential for Cl practically abolished the niflumic acid response. The niflumic acid results suggest that certain NSAIDs can directly alter Cl conductance in the bovine RPE, apparently independently of cyclooxygenase inhibition.