Cloning and function of the rat colonic epithelial K+ channel K(V)LQT1

K(V)LQT1 (K(V)LQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of K(V)LQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to K(V)LQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of K(V)LQT1 in crypt cells and surface epithelium. Expression of rK(V)LQT1 in Xenopus oocytes induced a typical delayed activated K+ current. that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that K(V)QT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.

The human temporal cortex: Characterization of neurons expressing nitric oxide synthase, neuropeptides and calcium-binding proteins, and their glutamate receptor subunit profiles

Immunocytochemical techniques were used to examine the distribution of neurons immunoreactive (-ir) for nitric oxide synthase (nNOS), somatostatin (SOM), neuropeptide Y (NPY), parvalbumin (PV), calbindin (CB) and calretinin (CH), in the inferotemporal gyros (Brodmann's area 21) of the human neocortex. Neurons that colocalized either nNOS or SOM with PV, CB or CR were also identified by double-labeling techniques. Furthermore, glutamate receptor subunit profiles (GluR1, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1) were also determined for these cells. The number and distribution of cells containing nNOS, SOM, NPY, PV, CB or CR differed for each antigen. In addition, distinct subpopulations of neurons displayed different degrees of colocalization of these antigens depending on which antigens were compared. Moreover, cells that contained nNOS, SOM, NPY, PV, GB or CR expressed different receptor subunit profiles. These results show that specific subpopulations of neurochemically identified nonpyramidal cells may be activated via different receptor subtypes. As these different subpopulations of cells project to specific regions of pyramidal calls, facilitation of subsets of these cells via different receptor subunits may activate different inhibitory circuits. Thus, various distinct, but overlapping, inhibitory circuits may act in concert in the modulation of normal cortical function, plasticity and disease.

Murine ventricular L-type Ca2+ current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor

1 The functional coupling of B-2-adrenoceptors (beta (2)-ARs) to murine L-type Ca2+ current (I-Ca(L)) was investigated with two different approaches. The beta (2)-AR signalling cascade was activated either with the beta (2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta (2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta (2)-ARs). Ca2+ and Ba2+ currents were recorded in the whole-cell and cell-attached configuration of the patch- clamp technique, respectively. 2 Zinterol (10 muM) significantly increased I-Ca(L) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta (1)-AR subtype, since it was blocked by the beta (1)-AR selective antagonist CGP 20712A (300 nM). The beta (2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I-Ca(L) to zinterol. 3 In myocytes with beta (2)-AR overexpression I-Ca(L) was not stimulated by the activated signalling cascade. On the contrary, I-Ca(L) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I-Ca(L). The beta (2)-AR inverse agonist ICI 118,551 did not further decrease I-Ca(L). PTX-treatment increased current amplitude to values found in control myocytes. 4 In conclusion, there is no evidence for beta (2)-AR mediated increases of I-Ca(L) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta (2)-AR responses to zinterol, but augments beta (1)-AR mediated increases of I-Ca(L). In the mouse model of beta (2)-AR overexpression I-Ca(L) is reduced due to tonic activation of Gi-proteins.

Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line.

The plasma membrane Ca2+ pump is a key regulator of cytosolic free Ca2+. Recent studies have demonstrated the dynamic expression of the plasma membrane Ca2+ pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca2+ ATPase (PMCA1) isoform of the plasma membrane Ca2+ pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously. (C) 2001 Elsevier Science Inc. All rights reserved.

Self-reported calcium intake and bone mineral content in children and adolescents

Objective: We examined the relationship between self-reported calcium (Cal intake and bone mineral content (BMC) in children and adolescents. We hypothesized that an expression of Ca adjusted for energy intake (El), i.e., Ca density, would be a better predictor of BMC than unadjusted Ca because of underreporting of EI. Methods: Data were obtained on dietary intakes (repeated 24-hour recalls) and BMC (by DEXA) in a cross-section of 227 children aged 8 to 17 years. Bivariate and multivariate analyses were used to examine die relationship between Ca, Ca density, and the dependent variables total body BMC and lumbar spine BMC. Covariates included were height, weight, bone area, maturity age, activity score and El. Results: Reported El compared to estimated basal metabolic rate suggested underreporting of El. Total body and lumbar spine BMC were significantly associated with El, but not Ca or Ca density, in bivariate analyses. After controlling for size and maturity, multiple linear regression analysis revealed unadjusted Ca to be a predictor of BMC in males in the total body (p = 0.08) and lumbar spine (p = 0.01). Unadjusted Ca was not a predictor of BMC at either site in females. Ca density was not a better predictor of BMC at either site in males or females. Conclusions: The relationship observed in male adolescents in this study between Ca intake and BMC is similar to that seen in clinical trials. Ca density did not enable us to see a relationship between Ca intake and BMC in females, which may reflect systematic reporting errors or that diet is not a limiting factor in this group of healthy adolescents.

The effect of channel geometry and wall boundary conditions on the formation of extrusion surface instabilities for LLDPE

It is believed that surface instabilities can occur during the extrusion of linear low density polyethylene due to high extensional stresses at the exit of the die. Local crack development can occur at a critical stress level when melt rupture is reached. This high extensional stress results from the rearrangement of the flow at the boundary transition between the wall exit and the free surface. The stress is highest at the extrudate surface and decreases into the bulk of the material. The location of the region where the critical level is reached can determine the amplitude of the extrudate surface distortion, This paper studies the effect of wall slip on the numerically simulated extensional stress level at the die exit and correlates this to the experimentally determined amplitude of the surface instability. The effect of die exit radius and die wall roughness on extrusion surface instabilities is also correlated to the exit stress level in the same way. Whereas full slip may completely suppress the surface instability, a reduction in the exit stress level and instability amplitude is also shown for a rounded die exit and a slight increase in instability is shown to result from a rough die wall. A surface instability map demonstrates how the shear rate for onset of extrusion surface instabilities can be predicted on the basis of melt strength measurements and simulated stress peaks at the exit of the die. (C) 2001 Elsevier Science B.V. All rights reserved.

On the nonlocality of the quantum channel in the standard teleportation protocol

By exhibiting a violation of a novel form of the Bell-CHSH inequality, Żukowski has recently established that the quantum correlations exploited in the standard perfect teleportation protocol cannot be recovered by any local hidden variables model. In the case of imperfect teleportation, we show that a violation of a generalized form of Żukowski's teleportation inequality can only occur if the channel state, considered by itself, already violates a Bell-CHSH inequality. On the other hand, the fact that the channel state violates a Bell-CHSH inequality is not sufficient to imply a violation of Żukowski's teleportation inequality (or any of its generalizations). The implication does hold, however, if the fidelity of the teleportation exceeds ≈ 0.90. © 2001 Elsevier Science B.V. All rights reserved.

A common inhibitory binding site for zinc and odorants at the voltage-gated K+ channel of rat olfactory receptor neurons

This study compared the effects of zinc and odorants on the voltage-gated K+ channel of rat olfactory neurons. Zinc reduced current magnitude, depolarized the voltage activation curve and slowed activation kinetics without affecting inactivation or deactivation kinetics. Zinc inhibition was potentiated by the NO compound, S-nitroso-cysteine. The pH- and diethylpyrocarbonate-dependence of zinc inhibition suggested that zinc acted by binding to histidine residues. Cysteine residues were eliminated as contributing to the zinc-binding site. The odorants, acetophenone and amyl acetate, also reduced current magnitude, depolarized the voltage activation curve and selectively slowed activation kinetics. Furthermore, the diethylpyrocarbonate- and pH-dependence of odorant inhibition implied that the odorants also bind to histidine residues. Zinc inhibitory potency was dramatically diminished in the presence of odorants, implying competition for a common binding site. These observations indicate that the odorants and zinc share a common inhibitory binding site on the external surface of the voltage-gated K+ channel.

The surface accessibility of the glycine receptor M2-M3 loop is increased in the channel open state

Mutations in the extracellular M2-M3 loop of the glycine receptor (GlyR) alpha1 subunit have been shown previously to affect channel gating. In this study, the substituted cysteine accessibility method was used to investigate whether a structural rearrangement of the M2-M3 loop accompanies GlyR activation. All residues from R271C to V277C were covalently modified by both positively charged methanethiosulfonate ethyltrimethylammonium (MTSET) and negatively charged methanethiosulfonate ethylsulfonate (MTSES), implying that these residues form an irregular surface loop. The MTSET modification rate of all residues from R271C to K276C was faster in the glycine-bound state than in the unliganded state. MTSES modification of A272C, L274C, and V277C was also faster in the glycine-bound state. These results demonstrate that the surface accessibility of the M2-M3 loop is increased as the channel transitions from the closed to the open state, implying that either the loop itself or an overlying domain moves during channel activation.

Ivermectin, an unconventional agonist of the glycine receptor chloride channel

The effects of the antihelmintic, ivermectin, were investigated in recombinantly expressed human alpha (1) homomeric and alpha (1)beta heteromeric glycine receptors (GlyRs), At low (0.03 muM) concentrations ivermectin potentiated the response to sub-saturating glycine concentrations, and at higher (greater than or equal to0.03 muM) concentrations it irreversibly activated both alpha (1) homomeric and alpha (1)beta heteromeric GlyRs. Relative to glycine-gated currents, ivermectin-gated currents exhibited a dramatically reduced sensitivity to inhibition by strychnine, picrotoxin, and zinc. The insensitivity to strychnine could not be explained by ivermectin preventing the access of strychnine to its binding site. Furthermore, the elimination of a known glycine- and strychnine-binding site by site-directed mutagenesis had little effect on ivermectin sensitivity, demonstrating that the ivermectin- and glycine-binding sites were not identical. Ivermectin strongly and irreversibly activated a fast-desensitizing mutant GlyR after it had been completely desensitized by a saturating concentration of glycine. Finally, a mutation known to impair dramatically the glycine signal transduction mechanism had little effect on the apparent affinity or efficacy of ivermectin, Together, these findings indicate that ivermectin activates the GlyR by a novel mechanism.

The influence of calcium on granular sludge in a full-scale UASB treating paper mill wastewater

Calcium precipitation can have a number of effects on the performance of high-rate anaerobic performance including cementing of the sludge bed, limiting diffusion, and diluting the active biomass. The aim of this study was to observe the influence of precipitation in a stable full-scale system fed with high-calcium paper factory wastewater. Granules were examined from an upflow anaerobic sludge blanket reactor (volume 1,805 m(3)) at a recycled paper mill with a loading rate of 5.7-6.6 kgCOD.m(-3).d(-1) and influent calcium concentration of 400-700 gCa(.)m(-3). The granules were relatively small (1 mm), with a 200-400 mum core of calcium precipitate as observed with energy dispersive X-ray spectroscopy. Compared to other granules, Methanomicrobiales not Methanobacteriales were the dominant hydrogen or formate utilisers, and putative acidogens were filamentous. The strength of the paper mill fed granules was very high when compared to granules from other full-scale reactors, and a partial linear correlation between granule strength and calcium concentration was identified.

New type of dual-channel PAM chlorophyll fluorometer for highly sensitive water toxicity biotests

A new type of dual-channel PAM chlorophyll fluorometer has been developed, which is specialised in the detection of extremely small differences in photosynthetic activity in algae or thylakoids suspensions. In conjunction with standardised algae cultures or isolated thylakoids, the new device provides an ultrasensitive biotest system for detection of toxic substances in water samples. In this report, major features of the new device are outlined and examples of its performance are presented using suspensions of Phaeodactylum tricornutum (diatoms) and of freeze-dried thylakoids of Lactuca sativa (salad). Investigated and reference samples are exposed to the same actinic intensity of pulse-modulated measuring light. The quantum yields are assessed by the saturation pulse method. Clock-triggered repetitive measurements of quantum yield typically display a standard deviation of 0.1%, corresponding to the inhibition induced by 0.02 mug diuron l(-1). Hence, for diuron or compounds with similar toxicity, the detection limit is well below the 0.1 mug l(-1) defined as the limit for the presence of a single toxic substance in water by the European Commission drinking water regulation. The amounts of water and biotest material required for analysis are very small, as a single assay involves two 1 ml samples, each containing ca. 0.5 mug chlorophyll. Both with Phaeodactylum and thylakoids the relationship between inhibition and diuron concentration is strictly linear up to 10% inhibition, with very similar slopes. Apparent inhibition depends on the actinic effect of the measuring light, showing optima at 6 and 4 mumol quanta m(-2) s(-1) with Phaeodactylum and thylakoids, respectively.