Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.     

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Confers Glibenclamide Sensitivity to Outwardly Rectifying Chloride Channel (ORCC) in Hi-5 Insect Cells

Increasing evidence is now accumulating for the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in the control of the outwardly rectifying chloride channel (ORCC). We have examined the sensitivity of ORCC to the sulfonylurea drug glibenclamide in Hi-5 (Trichoplusia ni) insect cells infected with recombinant baculovirus expressing either wild-type CFTR, ΔF508-CFTR or E. coliβ galactosidase cDNA and in control cells either infected with virus alone or uninfected. Iodide efflux and single channel patch-clamp experiments confirmed that forskolin and 1-methyl-3-isobutyl xanthine (IBMX) or 7-methyl-1,3 dipropyl xanthine (DPMX) activate CFTR channels (unitary conductance: 9.1 ± 1.6 pS) only in cells expressing CFTR. In contrast, we identified 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS)-sensitive ORCC in excised membrane patches in any of the cells studied, with similar conductance (22 ± 2.5 pS at −80 mV; 55 ± 4.1 pS at +80 mV) and properties. In the presence of 500 μm SITS, channel open probability (P _o ) of ORCC was reversibly reduced to 0.05 ± 0.01 in CFTR-cells, to 0.07 ± 0.02 in non-CFTR expressing cells and to 0.05 ± 0.02 in ΔF508-cells. In Hi-5 cells that did not express CFTR, glibenclamide failed to inhibit ORCC activity even at high concentrations (100 μm), whereas 500 μm SITS reversibly inhibited ORCC. In contrast in cells expressing CFTR or ΔF508, glibenclamide dose dependently (IC₅₀= 17 μm, Hill coefficient 1.2) and reversibly inhibited ORCC. Cytoplasmic application of 100 μm glibenclamide reversibly reduced P _o from 0.88 ± 0.03 to 0.09 ± 0.02 (wash: P _o = 0.85 ± 0.1) in CFTR cells and from 0.89 ± 0.05 to 0.08 ± 0.05 (wash: P _o = 0.87 ± 0.1) in ΔF508 cells. In non-CFTR expressing cells, glibenclamide (100 μm) was without effect on P _o (control: P _o = 0.89 ± 0.09, glib.: P _o = 0.86 ± 0.02; wash: P _o = 0.87 ± 0.05). These data strongly suggest that the expression of CFTR confers glibenclamide sensitivity to the ORCC in Hi-5 cells. Received: 23 October 1998/Revised: 29 December 1998     

On the stability of a model of testosterone dynamics

We prove the global asymptotic stability of a well-known delayed negative-feedback model of testosterone dynamics, which has been proposed as a model of oscillatory behavior. We establish stability (and hence the impossibility of oscillations) even in the presence of delays of arbitrary length.Supported in part by AFOSR Grant F49620-01-1-0063, NIH Grant P20 GM64375, and Dimacs.E.D. Sontag: Supported in part by AFOSR Grant F49620-01-1-0063 and NIH Grant R01 GM46383.Acknowledgement We would like to thank Augusto Ponce for useful suggestions.     

Phenazine-1-carboxylic acid is negatively regulated and pyoluteorin positively regulated by gacA in Pseudomonas sp. M18

The biosynthesis of antimicrobial metabolites is controlled by the GacS/GacA two-component regulatory system in Pseudomonas species. The production of phenazine-1-carboxylic acid and pyoluteorin is differentially regulated by GacA in Pseudomonas sp. M18. Pyoluteorin was reduced to nondetectable level in culture of the gacA insertional mutant strain M18G grown in King's medium B broth, whereas phenazine-1-carboxylic acid production was increased 30-fold over that of the wild-type strain. Production of both antibiotics was restored to wild-type levels after complementation in trans with the wild-type gacA gene. Expression of the translational fusions phzA'-'lacZ and pltA'-'lacZ confirmed the effect of GacA on both biosynthetic operons.     

Survey of the number of two-component response regulator genes in the complete and annotated genome sequences of prokaryotes

The numbers of potential response regulator genes were determined from the complete and annotated genome sequences of Archaea and Bacteria. The numbers of each class of response regulators are shown for each organism, determined principally from BLASTP searches, but with reference to the gene category lists where available. The survey shows that for Bacteria there is a link between the total number of potential response regulator genes and both the genome complexity (number of potential protein-coding genes) and the organism's lifestyle/habitat. Increasingly complex lifestyles and genome complexities are matched by an increase in the average number of potential response regulator genes per genome, indicating that a higher degree of complexity requires a higher level of control of gene expression and cellular activity. Detailed results of this study are available online at and.     

Plant two-component systems: principles,functions, complexity and cross talk

Two-component systems have emerged as important sensing/response mechanisms in higher plants. They are composed of hybrid histidine kinases, histidine-containing phosphotransfer domain proteins and response regulators that are biochemically linked by His-to-Asp phosphorelay. In plants two-component systems play a major role in cytokinin perception and signalling and contribute to ethylene signal transduction and osmosensing. Furthermore, developmental processes like megagametogenesis in Arabidopsis thaliana and flowering promotion in rice (Oryza sativa) involve elements of two-component systems. Two-component-like elements also function as components of the Arabidopsis circadian clock. Because of the molecular mode of signalling, plant two-component systems also appear to serve as intensive cross talk and signal integration machinery. In this review we summarize the present knowledge about the principles and functions of two-component systems in higher plants and address several critical points with respect to cross talk, signal integration and specificity.Abbreviations AHK Arabidopsis histidine kinase - AHP Arabidopsis histidine-containing phosphotransfer domain protein - APRR Arabidopsis pseudo response regulator - ARR Arabidopsis response regulator - CCT CONSTANS CONSTANS-like TOC1 - CKI Cytokinin insensitive - CRE Cytokinin response - CTR Constitutive triple response - Ehd Early heading date - EIN Ethylene insensitive - ERS Ethylene response sensor - ETR Ethylene resistant - GARP-motif Found in Golden2 of maize, Arabidopsis B-type response regulators and Chlamydomonas Psr1 - HPt Histidine-containing phosphotransfer domain - NLS Nuclear localization signal - phyB Phytochrome B - TCS Two-component signalling - TOC Timing of CAB (chlorophyll a/b-binding protein) expression - WOL Wooden leg     

Synthesis of [26,27-2H6]brassinosteroids from 23,24-bisnorcholenic acid methyl ester

A number of hexadeuterated brassinosteroids (BS) containing a hydroxy group at C-22 or a 22R,23R-diol function were prepared starting from 23,24-bisnorcholenic acid methyl ester for biosynthetic studies. Synthesis of the cyclic part was accomplished via the initial hydroboration-oxidation of Delta(5)-double bond. The key step in the synthesis of the side chain involved addition of (2S)-[3,4-(2)H(6)]2,3-dimethylbutylphenyl sulfone to the corresponding C-22 aldehydes.     

Approaches on vegetative propagation of difficult-to-root <Emphasis Type="Italic">Salix caprea</Emphasis>

In the framework of a willow rust research project, it was necessary to include vegetatively propagated plant material of selected sallow trees (Salix caprea L.) into biotests for identification of pathotypes. Since it was not possible to root sufficient clonal plants by conventional cutting propagation, the applicability of tissue culture methods was tested. From 10 selected donor trees of Salix caprea newly sprouted shoots were harvested and transferred to nutrient media after surface disinfection. The cultures were grown at 20--22 °C, illuminated with warm-white fluorescent tubes. The majority of shoot tips and nodal segments died during the first month, but only with nursery-grown plants this was caused by bacteria contamination. Two clones could be established easily on hormone-free medium. Five clones could be initiated only after repeated subcultures on various media variants. Three clones failed completely. Different basic media compositions were tested and Woody Plant Medium, supplemented with 0.1% activated charcoal, proved to be best for most of the sallow clones. Well developed rooted plantlets were used in vitro for microcutting propagation. The resulting plants were transferred to soil and could be included in the rust screening program after acclimatising. The applicability of micropropagation for selected Salix caprea donor trees was strongly depending on the genotype. But the comparison of results from microcuttings with conventional cutting propagation showed that these methods were successful for different clones each.