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pICln has been proposed to be the swelling-activated anion channel responsible for ICl, swell, or a channel regulator. We tested the anion channel hypothesis by reconstituting recombinant pICln into artificial and biological membranes. Single channels were observed when pICln was reconstituted into planar lipid bilayers. In the presence of symmetrical 300 mM KCl, the channels had a high open probability and a slope conductance of 48 pS, and were outwardly rectifying. Reduction of trans KCl to 50 mM shifted the reversal potential by −31.2 ± 0.06 mV, demonstrating that the channel is at least seven times more selective for cations than for anions. Consistent with this finding, channel conductance was unaffected by substitution of Cl− with glutamate, but was undetectable when K+ was replaced by N-methyl-d-glucamine. Reconstitution of pICln into liposomes increased 86Rb+ uptake by three- to fourfold, but had no effect on 36Cl− uptake. Phosphorylation of pICln with casein kinase II or mutation of G54, G56, and G58 to alanine decreased channel open probability and 86Rb+ uptake. When added to the external medium bathing Sf9 cells, pICln inserted into the plasma membrane and increased cell cation permeability. Taken together, these observations demonstrate that channel activity is due to pICln and not minor contaminant proteins. However, these findings do not support the hypothesis that pICln is the anion-selective ICl, swell channel. The observed cation channel activity may reflect an as yet to be defined physiological function of pICln, or may be a consequence of in vitro reconstitution of purified, recombinant protein.

pICln is found ubiquitously in mammalian cells and is postulated to play a critical role in cell volume regulation. Mutagenesis studies led to the proposal that pICln is a swelling-activated anion channel. However, recent studies in Madin-Darby canine kidney cells and endothelial cells have shown that the protein is localized primarily to the cytoplasm. It has therefore been postulated that activation involves reversible translocation of pICln from the cytoplasm and insertion into the plasma membrane. We tested this hypothesis using several different approaches. Fractionation of C6 glioma cells into plasma membrane- and cytoplasm-containing fractions demonstrated that ∼90% of the recovered pICln was confined to the cytosol. Swelling had no effect on the relative amount of protein present in the plasma membrane fraction. Immunofluorescence microscopy revealed that pICln is localized primarily, if not exclusively, to the cytoplasm of swollen and nonswollen cells. Similarly, transfection of cells with a green fluorescent protein-labeled pIClnconstruct failed to reveal any membrane localization of the protein. These findings do not support the hypothesis that pICln is a volume regulatory anion channel activated by swelling-induced membrane insertion.

Introduction Lectures are an efficient and commonly used way of teaching large groups of students. Unfortunately such large groups may not lend themselves to much interaction, limiting and reducing the impact of the teaching material. In particular, anecdotal experience suggests that students are reluctant to ask questions pertaining to lectures when large numbers of other students are present. We implemented a novel method of taking questions during the ‘Neurology Week’ for students in their third year of the undergraduate medical degree course at the University of Glasgow; live text messaging. Methods In the introduction to half of the week9s didactic lectures, students were given a mobile phone number and encouraged to submit ‘live’ text questions to the speakers. A chairperson (EN or KD) would read texts as they arrived and either immediately ask the question to the speaker via a roving microphone or postpone the question until the end of the talk/section. The number and nature of the questions were compared in those sessions with and without the live text facility. Students and speakers took part in a survey to evaluate their experience of the ‘live text’ experiment. Results In 4 hours of lectures with text facility, 86 texts were received; these comprised 56 requesting factual clarification or expansion, 28 jokes, and 1 practical point about the lecture hall. Additionally, 2 oral questions were posed after the lectures. After 4 hours of lectures without text facility, there were 9 questions posed to the individual speakers, all requesting factual clarification. These oral questions and their answers were not shared with the rest of the group. After exclusion of jokes, the number of questions posed to lecturers where live texting was offered was found to be significantly greater than the number posed where no live texting was available (p=0.03, Mann Whitney U test). Students9 and speakers9 survey responses confirmed that they found the text–questioning to be enjoyable and worthwhile. Conclusions Considered use of cheap and readily available technology allows interactive teaching to take place even during large group lectures. Students ask more questions, and this ‘extra’ education is shared with the entire group. We believe that such methods should be utilised more widely and would be equally valuable during postgraduate teaching.

Exposure of cortical astrocytes to 325, 350, or 390 mosM culture media for 48 h caused a 1.4-, 2.1-, and 3.5-fold increase, respectively, in cellular content of the compatible osmolyte myo-inositol. Elevated myo-inositol levels accounted for approximately 56-100% of the solute needed by the cells for complete volume regulation under hypertonic conditions. Myo-inositol accumulation was associated with 4-5-fold (peak rate) and 1.8-2-fold (steady-state rate) increases in the rate of Na(+)-dependent myo-inositol uptake when cells were acclimated to 390 mosM culture medium for 12 h or 24-96 h, respectively. When medium osmolality was elevated by 25 mosM, peak and steady-state increases in myo-inositol uptake of 1.7-fold and 1.3-fold, respectively, were observed. Exposure to 390 mosM medium for 12-48 h induced a 3-8-fold increase in cotransporter mRNA levels suggesting that the increase in myo-inositol uptake is brought about by increased cotransporter gene expression. Abrupt return of hypertonic cells to an isotonic medium induced a rapid increase in myo-inositol efflux and a return of cotransporter mRNA to control values in < 2 h. In contrast, the cotransporter remained fully activated at hypertonic levels for 16 h. Between 16-24 h after the transfer, the rate of myo-inositol uptake returned to control values. The remarkable sensitivity of the cotransporter to hypertonic stress indicates that upregulation of myo-inositol transport in glial cells is likely to occur in a variety of disease states that cause an elevation of plasma osmolality. Slow downregulation of the cotransporter may be responsible in part for the slow loss of myo-inositol and cerebral edema that occurs with too rapid correction of chronic plasma hypertonicity.

Rat C6 glioma cells chronically acclimated to hypertonic media accumulate large quantities of inositol. When returned to isotonic conditions, the cells swell and lose inositol slowly via a four- to fivefold increase in the rate of passive inositol efflux. The inositol efflux pathway is a Na(+)-independent transport mechanism with low affinity for inositol and is inhibited by quinidine, quinine, various anion transport blockers, and cis-unsaturated fatty acids. Ionomycin-induced elevation of intracellular Ca2+ (Ca2+i) had no effect on basal or swelling-induced inositol efflux. Inositol efflux was not inhibited by chelation of Ca2+i with 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid. In addition, Ca2+i measured with fura 2 did not change during cell swelling, indicating that increases in Ca2+i do not regulate inositol efflux. Exposure of C6 cells to 20 nM phorbol 12-myristate 13-acetate, 0.5 mM adenosine 3',5'-cyclic monophosphate (cAMP), or 50 microM forskolin had no effect on basal inositol efflux but stimulated swelling-induced inositol loss by 2.6-, 2.2-, and 3.4-fold, respectively. Exposure to the protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine or staurosporine or downregulation of protein kinase C (PKC) activity, however, had no inhibitory effect on inositol efflux, and cellular cAMP levels were not altered by cell swelling. Taken together, these results indicate that stimulation of PKC and protein kinase A modulates the activity of the efflux pathway but is not required for swelling-induced activation. Ketoconazole, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, and gossypol, inhibitors of lipoxygenase enzymes, blocked both basal and swelling-induced inositol efflux, suggesting indirectly that lipoxygenase metabolites may be responsible for swelling-induced activation of the efflux mechanism. The characteristics of inositol efflux in C6 cells are similar to those described for volume regulatory sorbitol and taurine efflux in a number of cell types, suggesting the existence of a common transport mechanism.

C. elegans has provided important insights into neuromuscular system function and development. However, the animal's small size limits access to individual neurons and muscle cells for physiological, biochemical, and molecular study. We describe here primary culture methods that allow C. elegans embryonic cells to differentiate into neurons and muscle cells in vitro. Morphological, electrophysiological, and GFP reporter studies demonstrate that the differentiation and functional properties of cultured cells are similar to those observed in vivo. Enriched populations of cells expressing specific GFP reporters can be generated by fluorescence-activated cell sorting. Addition of double-stranded RNA to the culture medium induces dramatic knockdown of targeted gene expression. Primary nematode cell culture provides a new foundation for a wide variety of experimental opportunities heretofore unavailable in the field.

48 children who were educably mentally handicapped, learning disabled, achievers, and academically gifted took the Piers-Harris self-concept scale. Differences between groups on the 6 factors of the scale were examined. Achievers and gifted students had higher and more positive responses to self-concept statements about school ability and achievement than the other two groups.