Structural and biophysical properties of a synthetic channel-forming peptide: designing a clinically relevant anion selective pore.

The design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the cys-loop superfamily of ligand-gated ion channels are part of an ongoing research focus. Over 300 different sequences have been prepared based on the M2 transmembrane segment of the spinal cord glycine receptor α-subunit. A number of these sequences are water-soluble monomers that readily insert into biological membranes where they undergo supramolecular assembly, yielding channels with a range of selectivities and conductances. Selection of a sequence for further modifications to yield an optimal lead compound came down to a few key biophysical properties: low solution concentrations that yield channel activity, greater ensemble conductance, and enhanced ion selectivity. The sequence NK(4)-M2GlyR T19R, S22W (KKKKPARVGLGITTVLTMRTQW) addressed these criteria. The structure of this peptide has been analyzed by solution NMR as a monomer in detergent micelles, simulated as five-helix bundles in a membrane environment, modified by cysteine-scanning and studied for insertion efficiency in liposomes of selected lipid compositions. Taken together, these results define the structural and key biophysical properties of this sequence in a membrane. This model provides an initial scaffold from which rational substitutions can be proposed and tested to modulate anion selectivity. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Molecular and functional identification of organic anion transporter isoforms in cultured bovine mammary epithelial cells (BME-UV).

Mammary epithelial cells express a diversity of membrane transporters including members of organic cation and organic anion (OAT) transporter subfamilies. Four mammal OAT isoforms have been identified: OAT-1, OAT-2, OAT-3, and OAT-4. The pharmacological significance of OAT isoforms has been emphasized because of their role in the movement of a wide variety of substrates across epithelial barriers. The present study identified (molecularly and functionally) bovine OAT isoforms in bovine mammary epithelial (BME-UV) cells. mRNA expression levels of all tested transporters in BME-UV cells were less than expression levels of the corresponding transporters in bovine kidney. Directionality in the flux of P-aminohippuric acid and acetylsalicylate, compounds known to interact with OAT-1 and OAT-2, respectively, across BME-UV monolayers was not observed at the concentrations used in this study. Directionality was, however, observed in the flux of estrone sulfate (EsS). Adding probenecid, penicillin G or nonradiolabeled EsS to the apical donor compartment significantly increased the apical-to-basolateral flux of EsS across the BME-UV monolayer. These results suggest that BME-UV cells express an organic anion transport system, making it a potentially useful model to study the role of this transport system in the mammary epithelial barrier.

Cultured mammary epithelial monolayers (BME-UV) express functional organic anion and cation transporters.

There is ongoing concern about the potential adverse effects of xenobiotic residues in cows' milk to the human consumer. Although drugs that are intentionally administered to lactating dairy cattle are rigorously regulated to prevent harmful residues, there are numerous other potential sources of exposure that are not as easily controlled. For example, cattle may be exposed to mycotoxins, pesticides and/or persistent organic pollutants through feed, water and inhalation of polluted air. Accurate estimates of the rate and extent of excretion of these compounds into milk is important to assess the risk of exposure through cows' milk. In the present study, the expression of carrier mediated transport processes in cultured monolayers of an immortalized bovine mammary epithelial cell line (BME-UV) was determined using a flow-through diffusion cell system, selective substrates and inhibitors of organic cation transporters (OCT) and organic anion transporters (OAT). The basal-to-apical (BL-to-Ap) flux of tetraethylammonium and estrone sulfate significantly exceeded their flux in the opposite direction. The addition of selective inhibitors to the donor compartment significantly decreased the BL-to-Ap flux of either selective substrate. These results suggest that both OCT and OAT are functionally expressed by BME-UV cells.

Non-obstructive vas deferens and epididymis loss in cystic fibrosis rats.

This study utilizes morphological and mechanistic endpoints to characterize the onset of bilateral atresia of the vas deferens in a recently derived cystic fibrosis (CF) rat model. Embryonic reproductive structures, including Wolffian (mesonephric) duct, Mullerian (paramesonephric) duct, mesonephric tubules, and gonad, were shown to mature normally through late embryogenesis, with involution of the vas deferens and/or epididymis typically occurring between birth and postnatal day 4 (P4), although timing and degree of atresia varied. No evidence of mucus obstruction, which is associated with pathology in other CF-affected tissues, was observed at any embryological or postnatal time point. Reduced epididymal coiling was noted post-partum and appeared to coincide with, or predate, loss of more distal vas deferens structure. Remarkably, α smooth muscle actin expression in cells surrounding duct epithelia was markedly diminished in CF animals by P2.5 when compared to wild type counterparts, indicating reduced muscle development. RNA-seq and immunohistochemical analysis of affected tissues showed disruption of developmental signaling by Wnt and related pathways. The findings have relevance to vas deferens loss in humans with CF, where timing of ductular damage is not well characterized and underlying mechanisms are not understood. If vas deferens atresia in humans begins in late gestation and continues through early postnatal life, emerging modulator therapies given perinatally might preserve and enhance integrity of the reproductive tract, which is otherwise absent or deficient in 97% of males with cystic fibrosis.

Ibuprofen inhibits cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion.

We evaluated the acute effects of ibuprofen and salicylic acid on cAMP-mediated Cl- secretion (Isc) in both colonic and airway epithelia. In T84 cells, ibuprofen inhibited the forskolin-dependent Isc in a concentration-dependent manner, having an apparent Ki of 142 microM. Salicylic acid inhibited Isc with an apparent Ki of 646 microM. We determined whether ibuprofen would also inhibit the forskolin-stimulated Isc in primary cultures of mouse trachea epithelia (MTE) and human bronchial epithelia (HBE). Similar to our results in T84 cells, ibuprofen (500 microM) inhibited the forskolin-induced Isc in MTEs and HBEs by 59+/-4% (n = 11) and 39+/-6% (n = 8), respectively. Nystatin was employed to selectively permeabilize the basolateral or apical membrane to determine the effect of ibuprofen on apical Cl- (ICl) and basolateral K+ (IK) currents after stimulation by forskolin. After forskolin stimulation, ibuprofen (500 microM) reduced both the ICl and IK; reducing ICl and IK by 60 and 15%, respectively. To determine whether this inhibition of ICl was due to the inhibition of CFTR, the effects of ibuprofen and salicylic acid on CFTR Cl- channels in excised, inside-out patches from L-cells were evaluated. Ibuprofen (300 microM) reduced CFTR Cl- current by 60+/-16% and this was explained by a short-lived block (approximately 1.2 ms) which causes an apparent reduction in single channel amplitude from 1.07+/-0.04 pA to 0.59+/-0.04 pA (n = 3). Similarly, salicylic acid (3 mM) reduced CFTR Cl- current by 50+/-8% with an apparent reduction in single channel amplitude from 1.08+/-0.03 pA to 0.48+/-0.06 pA (n = 4). Based on these results, we conclude that the NSAIDs ibuprofen and salicylic acid inhibit cAMP-mediated Cl- secretion in human colonic and airway epithelia via a direct inhibition of CFTR Cl- channels as well as basolateral membrane K+ channels. This may reduce their efficacy in conjunction with other therapeutic strategies designed to increase CFTR expression and/or function in secretory epithelia.

Observation of a topologically non-trivial surface state in half-Heusler PtLuSb (001) thin films.

The discovery of topological insulators, materials with bulk band gaps and protected cross-gap surface states in compounds such as Bi2Se3, has generated much interest in identifying topological surface states (TSSs) in other classes of materials. In particular, recent theoretical calculations suggest that TSSs may be found in half-Heusler ternary compounds. If experimentally realizable, this would provide a materials platform for entirely new heterostructure spintronic devices that make use of the structurally identical but electronically varied nature of Heusler compounds. Here we show the presence of a TSS in epitaxially grown thin films of the half-Heusler compound PtLuSb. Spin- and angle-resolved photoemission spectroscopy, complemented by theoretical calculations, reveals a surface state with linear dispersion and a helical tangential spin texture consistent with previous predictions. This experimental verification of topological behaviour is a significant step forward in establishing half-Heusler compounds as a viable material system for future spintronic devices.

ATP alters current fluctuations of cystic fibrosis transmembrane conductance regulator: evidence for a three-state activation mechanism.

The cystic fibrosis gene product cystic fibrosis transmembrane conductance regulator (CFTR) is a low conductance, cAMP-regulated Cl- channel. Removal of cytosolic ATP causes a cessation of cAMP-dependent kinase-phosphorylated CFTR channel activity that resumes upon ATP addition. (Anderson, M. P., H. A. Berger, D. R. Rich, R. J. Gregory, A. E. Smith, and M. J. Welsh. 1991. Cell. 67:775-784). The aim of this study was to quantify possible effects of ATP on CFTR gating. We analyzed multichannel records since only 1 of 64 patches contained a single channel. ATP increased the channel open probability (Po) as a simple Michaelis-Menten function of concentration; the effect was half maximal at 24 microM, reached a maximum of 0.44, and had a Hill coefficient of 1.13. Since the maximum Po was not 1, the simplest description of the effect of ATP on CFTR gating is the noncooperative three-state mechanism of del Castillo and Katz (1957. Proceedings of the Royal Society of London. B. 146:369-381). We analyzed current fluctuations to quantify possible changes in CFTR gating. The power density spectra appeared to contain a single Lorentzian in the range of 0.096-31 Hz. Analysis of the corner frequency (fc) of this Lorentzian revealed that ATP increased 2 pi fc as a Michaelis-Menten function with a Hill coefficient of 1.08, and it provided estimates of the ATP dissociation constant (44 tau open (154 ms), and the ATP-sensitive tau close [(185 ms) (44 microM/[ATP] + 1)]. These results suggest that the binding reaction is rapid compared to the opening and closing rates. Assuming that there is a single set of closed-to-open transitions, it is possible to verify the outcome of fluctuation analysis by comparing fluctuation-derived estimates of Po with measures of Po from current records. The two values were nearly identical. Thus, noise analysis provides a quantitative description of the effect of ATP on CFTR opening. The noncooperative three-state model should serve as a basis to understand possible alterations in CFTR gating resulting from regulators or point mutations.

Regulation of CFTR Cl- channel gating by ADP and ATP analogues.

The cystic fibrosis gene product (CFTR) is a chloride channel which, once phosphorylated, is regulated by nucleotide phosphates (Anderson, M. P., and M. J. Welsh. 1992. Science. 257:1701-1704; Venglarik, C. J., B. D. Schultz, R. A. Frizzell, and R. J. Bridges. 1994. Journal of General Physiology. 104:123-146). Nucleotide triphosphates initiate channel activity, while nucleotide diphosphates and nonhydrolyzable ATP analogues do not. To further characterize the role of these compounds on CFTR channel activity we examined their effects on chloride channel currents in excised inside-out membrane patches from CFTR transfected mouse L cells. ADP competitively inhibited ATP-dependent CFTR channel gating with a Ki of 16 +/- 9 microM. AMP neither initiated CFTR channel gating nor inhibited ATP-dependent CFTR channel gating. Similarly, ATP analogues with substitutions in the phosphate chain, including AMPCPP, AMPPCP, AMPPNP, and ATP gamma S failed to support CFTR channel activity when present at the cytoplasmic face of the membrane and none of these analogues, when present at three to 10-fold excess of ATP, detectably altered ATP-dependent CFTR channel gating. These data suggest that none of these ATP analogues interact with the ATP regulatory site of CFTR which we previously characterized and, therefore, no inference regarding a requirement for ATP hydrolysis in CFTR channel gating can be made from their failure to support channel activity. Furthermore, the data indicate that this nucleotide regulatory site is exquisitely sensitive to alterations in the phosphate chain of the nucleotide; only a nonsubstituted nucleotide di- or triphosphate interacts with this regulatory site. Alternative recording conditions, such as the presence of kinase and a reduction in temperature to 25 degrees C, result in a previously uncharacterized kinetic state of CFTR which may exhibit distinctly different nucleotide dependencies.

Influence of market stress and protein level on feeder pig hematologic and blood chemical values.

One hundred twenty crossbred feeder pigs were used in 2 trials to determine the effects of food and water deprivation at the auction market and the effects of protein levels of receiving diet on blood chemical values. Food- and water-deprived animals had significantly higher packed-cell volume, colloid osmotic pressure, and cortisol values than did nondeprived animals. Total osmolality and plasma triiodothyronine values were significantly lower in deprived animals. Measurable effects of food and water deprivation were no longer apparent by 14 days after arrival at the research facility. Plasma colloid osmotic pressure had a positive linear relationship with increasing dietary protein level and was statistically different among levels of protein fed. Gilts had higher plasma triiodothyronine values than did barrows. Differential WBC ratios were not different between groups. Measurable differences for treatments (food and water deprivation vs food and water access; and level of protein in the receiving diet) were no longer apparent 84 days after pigs had arrived at the finishing unit.

Serum chemical profile of feeder pigs, as influenced by market stress and feeding regimen.

Two hundred eighty-eight crossbred feeder pigs were used in 2 trials to determine the effects of feed and/or water deprivation at an auction market, and the effects of restricting the intake of the receiving diet on their serum chemical profile. The study also was designed to assess the value of the serum chemical profile as a diagnostic data base for stress disorders in feeder pigs. Performance data indicated that feeder pigs provided water only at the auction facilities lost significantly more weight than did those provided feed and water. Feeder pigs deprived of both feed and water were not significantly different in body weight from either group. Several serum chemical values (creatinine, triglycerides, cholesterol, blood urea nitrogen, and lactate dehydrogenase) were significantly influenced by feed deprivation, but not by feed and water deprivation. However, only the serum creatinine values were significantly different after the 24-hour posttransport period. There were no significant differences in pig weight or serum chemical values 84 days after pigs had arrived at the finishing unit. The serum chemical profile, widely used in human medicine, appears not to provide a reliable marker for identification of short-term nutritional deprivation, nor for transport stress in feeder pigs.

Estimated change in blood lead concentration in control populations.

Investigators have conducted several studies to assess the effectiveness of lead hazard interventions in reducing children's blood lead levels. For practical and ethical reasons, many of these studies have not included control populations. It is, therefore, impossible for researchers to determine to what extent the reported decline in blood lead concentrations has resulted from intervention versus other factors. In the current retrospective analysis, the authors estimated the change in children's blood lead levels in control populations at 12-mo follow-up. The results suggest that an average 9% decline may be attributed to factors that are unrelated to intervention. Declines of approximately 25% have been reported following several lead-hazard interventions. For these studies, the results of the authors' analysis suggest that approximately 16% of the decline is attributed directly to the intervention.

Epinephrine effects on gastrin and gastric secretions in normal and stress-susceptible pigs and in dogs.

1. Epinephrine induced plasma gastrin concentration increased linearly in dogs, and significantly in pigs. 2. Epinephrine induced gastric secretion rate increased in dogs and decreased in pigs, and yet significantly reduced gastric pH only in dogs. 3. Statistical differences in heart and respiratory rate with epinephrine versus saline infusion were detected only in pigs. 4. Stress-susceptible pigs had a significant decrease in gastric secretion pH, heart rate and increased respiration compared to normal pigs, during saline infusion.

Lack of conventional ATPase properties in CFTR chloride channel gating.

CFTR shares structural homology with the ABC transporter superfamily of proteins which hydrolyze ATP to effect the transport of compounds across cell membranes. Some superfamily members are characterized as P-type ATPases because ATP-dependent transport is sensitive to the presence of vanadate. It has been widely postulated that CFTR hydrolyzes ATP to gate its chloride channel. However, direct evidence of CFTR hydrolytic activity in channel gating is lacking and existing circumstantial evidence is contradictory. Therefore, we evaluated CFTR chloride channel activity under conditions known to inhibit the activity of ATPases; i.e., in the absence of divalent cations and in the presence of a variety of ATPase inhibitors. Removal of the cytosolic cofactor, Mg2+, reduced both the opening and closing rates of CFTR suggesting that Mg2+ plays a modulatory role in channel gating. However, channels continued to both open and close showing that Mg2+ is not an absolute requirement for channel activity. The nonselective P-type ATPase inhibitor, vanadate, did not alter the gating of CFTR when used at concentrations which completely inhibit the activity of other ABC transporters (1 mM). Higher concentrations of vanadate (10 mM) blocked the closing of CFTR, but did not affect the opening of the channel. As expected, more selective P-type (Sch28080, ouabain), V-type (bafilomycin A1, SCN-) and F-type (oligomycin) ATPase inhibitors did not affect either the opening or closing of CFTR. Thus, CFTR does not share a pharmacological inhibition profile with other ATPases and channel gating occurs in the apparent absence of hydrolysis, although with altered kinetics. Vanadate inhibition of channel closure might suggest that a hydrolytic step is involved although the requirement for a high concentration raises the possibility of previously uncharacterized effects of this compound. Most conservatively, the requirement for high concentrations of vanadate demonstrates that the binding site for this transition state analogue is considerably different than that of other ABC transporters.

Rescue of dysfunctional deltaF508-CFTR chloride channel activity by IBMX.

Nucleotide-dependent gating of DeltaF508-CFTR was evaluated in membrane patches excised from HEK 293 and mouse L-cells and compared to observations on wt-CFTR channels recorded in the same expression systems. DeltaF508-CFTR exhibited PKA activated, ATP-dependent channel gating. When compared to wt-CFTR, the Km for ATP was increased by ninefold (260 micron vs. 28 micron) and maximal open probability (Po) was reduced by 49% (0.21 +/- 0.06 vs. 0.41 +/- 0. 02). Additionally, in the absence of PKA, DeltaF508-CFTR inactivated over a 1 to 5 min period whereas wt-CFTR remained active. Time-dependent inactivation could be mimicked in wt-CFTR by the intermittent absence of ATP in the cytosolic solution. The effects of 3-isobutyl-1-methyl xanthine (IBMX), a compound reported to stimulate DeltaF508-CFTR, were evaluated on wt- and DeltaF508-CFTR channels. At concentrations up to 5 mm, IBMX caused a concentration dependent reduction in the observed single channel amplitude (i) of wt-CFTR (maximal observed reduction 35 +/- 3%). However, IBMX failed to significantly alter total patch current because of a concomitant 30% increase in Po. The effects of IBMX on DeltaF508-CFTR were similar to effects on wt-CFTR in that i was reduced and Po was increased by similar magnitudes. Additionally, DeltaF508-CFTR channel inactivation was dramatically slowed by IBMX. These results suggest that IBMX interacts with the ATP-bound open state of CFTR to introduce a short-lived nonconducting state which prolongs burst duration and reduces apparent single channel amplitude. A secondary effect observed in DeltaF508-CFTR, which may result from this interaction, is a prolongation of the activated state. In light of previously proposed linear kinetic models of CFTR gating, these results suggest that IBMX traps CFTR in an ATP-bound state which may preclude inactivation of DeltaF508-CFTR.

Interpretation of airborne asbestos measurements.

Transmission electron microscopy (TEM) is the preferred method of measuring airborne asbestos in buildings, but TEM measurements cannot be used directly in the existing equations relating risk to exposure because the equations are based on measurements made with a different technique--phase contrast microscopy (PCM). Comparison between measurements made by different methods is not simple because the methods differ in the size of particles they can detect, and the relationship between exposure and disease is thought to depend on, among other things, asbestos fiber size. Previous suggestions for converting TEM measurements to PCM equivalents lack generality because they fail to take into account the size distribution of the asbestos particles and the expectation that fiber-size distributions in current nonoccupational environments could differ from the workplaces of the past on which the risk equations are based. A mathematical model is presented for investigating the conversion of airborne asbestos measurements made by one method to an equivalent measurement made by another method. "Equivalent" means having the same potential to cause disease. The model clarifies the issues of concern and suggests approaches for obtaining meaningful conversion factors that will allow TEM measurements to be used in PCM-based risk equations.

Neurotransmitter-stimulated ion transport by cultured porcine vas deferens epithelium.

A collagenase-based dissociation technique has been developed to routinely establish monolayer cultures of freshly isolated porcine vas deferens epithelium. Cells isolated from each tissue are transferred to 25-cm(2) tissue culture flasks and grown in a standard cell culture medium. Flasks reach confluency in 3-4 days, and cells are subsequently seeded onto permeable supports. Cultured cells display a monolayer cobblestone appearance and are immunoreactive to anti-ZO-1 and anti-cytokeratin antibodies. Electron microscopy is employed to demonstrate the presence of junctional complexes and microvilli. When evaluated in modified Ussing chambers, cultured monolayers exhibit a basal lumen negative potential difference, high electrical resistance (>1,000 Omega. cm(2)), and respond to norepinephrine, vasopressin, ATP, adenosine, and histamine, with changes in short-circuit current indicative of anion secretion. Responses are significantly attenuated in Cl(-)- and/or HCO-free solutions. Attempts to further optimize culture conditions have shown that chronic exposure to insulin increases proliferation rates. Thus the culture method described will reliably produce viable neurotransmitter-responsive cell monolayers that will allow for the characterization of vas deferens epithelial function and associated control mechanisms.

Neurotransmitter-stimulated ion transport across cultured bovine mammary epithelial cell monolayers.

Bovine mammary epithelial (BME-UV) and myoepithelial (BMM-UV) cell lines were acquired with the goal of developing an in vitro model of mammary epithelia for the study of ion transport. The bovine mammary cell lines were successfully cultured on commercially available permeable supports, and results suggest that mammary epithelial cells, but not myoepithelial cells, form tight junctions necessary to perform a barrier function. Electrogenic ion transport was not observed in basal conditions. Acute exposure to norepinephrine or forskolin caused prototypic increases in short circuit current accompanied by a reduction in transmural resistance indicative of anion secretion through a conductive pathway. Bumetanide and N-(4-methyphenylsulfonyl)-N'-(4-trifluoro-methylphenyl)urea, inhibitors of Na+/K+/Cl- cotransport and cystic fibrosis transmembrane conductance anion channels, respectively, reduced forskolin-stimulated ion transport. Amiloride, an inhibitor of epithelial sodium channels, had no effect on basal or forskolin-stimulated ion transport. However, naturally occurring and synthetic corticosteroids induced the expression of amiloride sensitive current indicative of sodium absorption. Chronic exposure to increased apical ionic strength and/or reduced carbohydrate concentration were associated with reduced transepithelial resistance although forskolin-stimulated ion transport was unaffected. These results demonstrate that neurotransmitters and steroid hormones act directly on bovine mammary epithelial cells to acutely and chronically modulate the volume and composition of their secretions. The in vitro system that we describe can be further exploited to characterize cellular and molecular mechanisms associated with mammary function in health and disease.

Inhibition of enterotoxin-induced porcine colonic secretion by diarylsulfonylureas in vitro.

Muscle-stripped piglet colon was used to evaluate changes in short-circuit current (I(sc)) as an indicator of anion secretion. Mucosal exposure to Escherichia coli heat-stable (STa) or heat-labile enterotoxins (LT) stimulated I(sc) by 32 +/- 5 and 42 +/- 7 microA/cm(2), respectively. Enterotoxin-stimulated I(sc) was not significantly affected by either 4,4'-diaminostilbene-2, 2'-disulfonic acid or CdCl(2), inhibitors of Ca(2+)-activated Cl(-) channels and ClC-2 channels, respectively. Alternatively, N-(4-methylphenylsulfonyl)-N'-(4-trifluoromethylphenyl)urea (DASU-02), a compound that inhibits cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion, reduced I(sc) by 29 +/- 7 and 34 +/- 11 microA/cm(2), respectively. Two additional diarylsulfonylurea (DASU)-based compounds were evaluated for their effects on enterotoxin-stimulated secretion. The rank order of potency for inhibition by these three closely related DASU structures was identical to that observed for human CFTR. The degree of inhibition by each of these compounds was similar for both STa and LT. The structure- and concentration-dependent inhibition shown indicates that CFTR mediates both STa- and LT-stimulated colonic secretion. Similar structure-dependent inhibitory effects were observed in forskolin-stimulated rat colonic epithelium. Thus DASUs compose a family of inhibitors that may be of therapeutic value for the symptomatic treatment of diarrhea resulting from a broad spectrum of causative agents across species.

Dynamic nuclear polarization by electrical spin injection in ferromagnet-semiconductor heterostructures.

Electrical spin injection from Fe into AlxGa1-xAs quantum well heterostructures is demonstrated in small (<500 Oe) in-plane magnetic fields. The measurement is sensitive only to the component of the spin that precesses about the internal magnetic field in the semiconductor. This field is much larger than the applied field and depends strongly on the injection current density. Details of the observed hysteresis in the spin injection signal are reproduced in a model that incorporates the magnetocrystalline anisotropy of the epitaxial Fe film, spin relaxation in the semiconductor, and the dynamic polarization of nuclei by the injected spins.