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Author(s): David N. Sheppard, Anthony P. Davis Author Affiliations: Pore-forming small molecules offer a promising way to tackle cystic fibrosis In cystic fibrosis, abnormalities in the ion-channel protein CFTR cause [...], In cystic fibrosis, ion-transport abnormalities cause problems in many organs. A small molecule that forms cell-membrane pores allowing ion transport shows therapeutic promise in human cells and a model of the disease. A natural molecule that facilitates ion transport aids lung function.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a [Cl.sup.-] channel gated by ATP-driven nucleotide-binding domain (NBD) dimerization. Here we exploit species differences between human and murine CFTR to investigate CFTR channel gating. Using homologous recombination, we constructed human-murine CFTR (hmCFTR) chimeras with sequences from NBD1, NBD2, or the regulatory domain (RD) of human CFTR replaced by the equivalent regions of murine CFTR. The gating behavior of hmRD and human CFTR were indistinguishable, whereas hmNBD1 and hmNBD2 had subtle effects on channel gating, prolonging both burst duration and interburst interval. By contrast, hmNBD1+2, containing both NBDs of murine CFTR, reproduced the gating behavior of the subconductance state of murine CFTR, which has dramatically prolonged channel openings. The CFTR potentiator pyrophosphate (PPi) enhanced human, hmRD, and hmNBD1 CFTR [Cl.sup.-] currents, but not those of hmNBD2, hmNBD1+2, and murine CFTR. By analyzing the rate-equilibrium free-energy relationships of chimeric channels, we obtained snapshots of the conformation of the NBDs during ATP-driven dimerization. Our data demonstrate that the conformation of NBD1 changes before that of NBD2 during channel opening. This finding suggests that NBD dimerization does not proceed by a symmetric tweezer-like motion, but instead in an asymmetric fashion led by NBD1. We conclude that the NBDs of murine CFTR determine the unique gating behavior of its subconductance state, whereas NBD2 controls channel potentiation by [PP.sub.i]. ATP-binding cassette transporter | chloride ion channel | cystic fibrosis | recombinational cloning | rate-equilibrium free-energy relationships

The revertant mutations G550E and 4RK [the simultaneous mutation of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K, and R766K] rescue the cell surface expression and function of F508del-cystic fibrosis (CF) transmembrane conductance regulator (-CFTR), the most common CF mutation. Here, we investigate their mechanism of action by using biochemical and functional assays to examine their effects on F508del and three CF mutations (R560T, A561E, and V5621) located within a conserved region of the first nucleotide-binding domain (NBD1) of CFTR. Like F508del, R560T and A561E disrupt CFTR trafficking. G550E rescued the trafficking defect of A561E but not that of R560T. Of note, the processing and function of V5621 were equivalent to that of wild-type (wt)-CFTR, suggesting that V5621 is not a disease-causing mutation. Biochemical studies revealed that 4RK generates higher steady-state levels of mature CFTR (band C) for wt-and V5621-CFTR than does G550E. Moreover, functional studies showed that the revertants rescue the gating defect of F508del-CFTR with different efficacies. 4RK modestly increased F508del-CFTR activity by prolonging channel openings, whereas G550E restored F508del-CFTR activity to wt levels by altering the duration of channel openings and closings. Thus, our data suggest that the revertants G550E and 4RK might rescue F508del-CFTR by distinct mechanisms. G550E likely alters the conformation of NBD1, whereas 4RK allows F508del-CFTR to escape endoplasmic reticulum retention/retrieval mediated by AFTs. We propose that AFTs might constitute a checkpoint for endoplasmic reticulum quality control. endoplasmic reticulum quality control | folding | membrane traffic | arginine-framed motifs | trafficking signals

Adenosylcobalamin (Ado-[B.sub.12]) is both the cofactor and inducer of ethanolamine ammonia lyase (EA-lyase), a catabolic enzyme for ethanolamine. De novo synthesis of Ado-[B.sub.12] by Salmonella enterica occurs only under anaerobic conditions. Therefore, aerobic growth on ethanolamine requires import of Ado-[B.sub.12] or a precursor (CN-[B.sub.12] or OH-[B.sub.12]) that can be adenosylated internally. Several known enzymes adenosylate corrinoids. The CobA enzyme transfers adenosine from ATP to a biosynthetic intermediate in de novo [B.sub.12] synthesis and to imported CN-[B.sub.12], OH-[B.sub.12], or Cbi (a [B.sub.12] precursor). The PduO adenosyl transferase is encoded in an operon (pdu) for cobalamin-dependent propanediol degradation and is induced by propanediol. Evidence is presented here that a third transferase (EutT) is encoded within the operon for ethanolamine utilization (eut). Surprisingly, these three transferases share no apparent sequence similarity. CobA produces sufficient Ado-[B.sub.12] to initiate eut operon induction and to serve as a cofactor for EA-lyase when [B.sub.12] levels are high. Once the eut operon is induced, the EutT transferase supplies more Ado-[B.sub.12] during the period of high demand. Another protein encoded in the operon (EutA) protects EA-lyase from inhibition by CN-[B.sub.12] but does so without adenosylation of this corrinoid.

When excised inside-out membrane patches are bathed in symmetrical [Cl.sup.-]-rich solutions, the current-voltage (I-V) relationship of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) [Cl.sup.-] currents inwardly rectifies at large positive voltages. To investigate the mechanism of inward rectification, we studied CFTR [Cl.sup.-] channels in excised inside-out membrane patches from cells expressing wild-type human and murine CFTR using voltage-ramp and step protocols. Using a voltage-ramp protocol, the magnitude of human CFTR [Cl.sup.-] current at +100 mV was 74 [+ or -] 2% (n = 10) of that at -100 mV. This rectification of macroscopic CFTR [Cl.sup.-] current was reproduced in full by ensemble currents generated by averaging single-channel currents elicited by an identical voltage-ramp protocol. However, using a voltage-step protocol the single-channel current amplitude (i) of human CFTR at + 100 mV was 88 [+ or -] 2% (n = 10) of that at--100 mV. Based on these data, we hypothesized that voltage might alter the gating behavior of human CFTR. Using linear three-state kinetic schemes, we demonstrated that voltage has marked effects on channel gating. Membrane depolarization decreased both the duration of bursts and the interburst interval, but increased the duration of gaps within bursts. However, because the voltage dependencies of the different rate constants were in opposite directions, voltage was without large effect on the open probability ([P.sub.o]) of human CFTR. In contrast, the [P.sub.o] of murine CFTR was decreased markedly at positive voltages, suggesting that the rectification of murine CFTR is stronger than that of human CFTR. We conclude that inward rectification of CFTR is caused by a reduction in i and changes in gating kinetics. We suggest that inward rectification is an intrinsic property of the CFTR [Cl.sup.-] channel and not the result of pore block. KEY WORDS: ATP-binding cassette transporter * cystic fibrosis * chloride ion channel * channel gating * voltage dependence

Research describing an improved image restoration algorithm is presented. Using original and diffraction-limited image pairs test results show that the nonlinear algorithm produces super-resolution.

An improved version of an algorithm designed to perform image restoration through use of nonlinear interpolative vector quantization is discussed. Using lapped bocks in the decoding process is helpful. The algorithm has been trained on original and diffraction-limited image paris. The nonlinearity of the algorithm gives super-resolution in restored images.

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) C1- channel is associated with a wide spectrum of disease. In the search for modulators of CFTR, pharmacological agents that interact directly with the CFTR C1-channel have been identified. Some agents stimulate CFTR by interacting with the nucleotide-binding domains that control channel gating, whereas others inhibit CFTR by binding within the channel pore and preventing C1- permeation. Knowledge of the molecular pharmacology of CFTR might lead to new treatments for diseases caused by the dysfunction of CFTR.

Effective community involvement programs and programs for the conservation of protected areas should be combined for the simultaneous benefit of people living near a protected area and the area itself. Management of parks by non-governmental organizations or by the private sector will increase tourist revenue, but exploitation of the parks to create tourist attractions should not distract them from protecting the area. Revenue from tourism will benefit local communities and improve their standards of living.

A modular, UNIX/X-Windows/Sun SPARC-based data acquisition and analysis system provides NASA X-Ray Timing Explorer (XTE) Proportional Counter Array (PCA) scientists with near real-time access to space flight instrument ground test data. This system processes data directly from five large-area, xenon-filled low-background collimated proportional counters at rates up to 2 MB/s in a networked, real-time environment. Science events are sent serially from the proportional counter flight electronics and multiplexed on a custom-designed data acquisition board. Each event is tagged with a time stamp of 1 [[micro]second] resolution and a detector identifier. A commercial Direct Memory Access (DMA) board interfaces directly to the data acquisition board allowing asynchronous transfer of buffered data to software residing on a high performance workstation. A windows-based interface provides an operator with acquisition command and control functions to capture data through the DMA board. Data is collected, buffered, and archived to magnetic disk. Histogramming and statistical functions are provided by the software enabling real-time pulse height spectroscopy, rate analysis, and spectral analysis.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chlorine channel expressed in apical and basolateral membranes in infected Fischer rat thyroid (FRT) epithelial cells. The chlorine channel is regulated by adenosine 3',5'-cyclic monophosphate (cAMP) phosphorylation even at low CFTR expressions. FRT epethelial dysfunction caused by three mutants reduces the level of apical chlorine current.

Mutant D836X that encodes the amino-terminal region of the cystic fibrosis transmembrane conductance regulator (CFTR) with a membrane-spanning domain and a nucleotide binding domain NBD. The R Domain constitutes a controlled chloride channel with characteristics that are similar to that of the wild-type channel. Activity increased with phosphorylation but conductance did not require phosphorylation.

EutR, a regulatory protein, regulates the expression of the ethanolamine utilization (eut) operon, in the presence of ethanolamine and adenosyl-cobalamin (Ado-B12). EutR is present within eut operon, and this provides a balance in the struggle for small quantities of Ado-B12 between lyase and eutR. Simultaneous stimulation of lyase and Ado-B12 leads to sustained eut operon induction and lyase activity.

Missense mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) are associated with milder cystic fibrosis. Replacements of arginine with histidine at residue 117, tryptophan at residue 334 or proline at residue 347 affect basic amino acids located at the external end of the second and the sixth putative membrane-spanning sequences. It is reported that all three mutants are correctly processed and generate cyclic AMP-regulated Cl- currents when expressed in heterologous epithelial cells.

Zora Neale Hurston's 'Their Eyes Were Watching God' deals with protagonist Janie's restless pursuit of a godhead to give meaning and order to her life. As Janie moves forward she experiences god-constructs that bring her steadily closer to her true godhead. This Modernist theme reveals Sigmund Freud's powerful influence on Hurston. Janie at last finds her optimum god-construct, reflecting Hurston's belief that God will be different for each person.

Adenosine triphosphate (ATP) dependent-potassium channel inhibitors and activators were examined to evaluate their effect on cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel regulated by ATP. Of the inhibitors tested, glibenclamide was determined to be the most effective blocker of CFTR. Potassium channel activators, on the other hand, proved to be inhibitory on CFTR. The order of inhibitory effect is diazoxide < tolbutamide < minoxidil sulfate = BRL 38227 < glibenclamide.

Many law enforcement agencies and child protective service (CPS) units of public child welfare agencies across the nation are conducting joint investigations of reported child abuse. The impetus for this [...]

Davidson, Donald J., Fiona M. Kilanowski, Scott H. Randell, David N. Sheppard, and Julia R. Dorin. A primary culture model of differentiated murine tracheal epithelium. Am J Physiol Lung Cell Mol Physiol 279: L766-L778, 2000.--The goal of this study was to develop a primary culture model of differentiated murine tracheal epithelium. When grown on semipermeable membranes at an air interface, dissociated murine tracheal epithelial cells formed confluent polarized epithelia with high transepithelial resistances (~12 k[Omega] [multiplied by] [cm.sup.2]) that remained viable for up to 80 days. Immunohistochemistry and light and electron microscopy demonstrated that the cells were epithelial in nature (cytokeratin positive, vimentin and [Alpha]-smooth muscle actin negative) and differentiated to form ciliated and secretory cells from day 8 after seeding onward. With RT-PCR, expression of the cystic fibrosis transmembrane conductance regulator (Cftr) and murine [Beta]-defensin (Defb) genes was detected (Defb-1 was constitutively expressed, whereas Defb-2 expression was induced by exposure to lipopolysaccharide). Finally, Ussing chamber experiments demonstrated an electrophysiological profile compatible with functional amiloride-sensitive sodium channels and cAMP-stimulated CFTR chloride channels. These data indicate that primary cultures of murine tracheal epithelium have many characteristics similar to those of murine tracheal epithelium in vivo. This method will facilitate the establishment of primary cultures of airway epithelium from transgenic mouse models of human diseases. cystic fibrosis; cystic fibrosis transmembrane conductance regulator; defensins; airway surface liquid; airway epithelium

I. INTRODUCTION A. Background Cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-dependent epithelial [Cl.sup.-] channel. It is located primarily in the apical membrane, where it provides a pathway for [...]

Research was conducted to examine an alternative role for the conserved aspartate (Asp) residue in phosphoryl transfer reactions that leads to alternative mechanisms for the mode of action of these important classes of enzymes based on the protonation states of the Asp and the phosphate group. Results indicate that the role of the Asp residue as a general acid or base in phosphoryl transfer reactions should be reconsidered. An alternative role of the Asp residue could be to stabilize the protonated forms of the phosphate species within the active site.

A technique for identifying image blur using vector quantizer encoder distortion is presented. This technique needs a set of training images blurred by each of the candidate blur functions to design a corresponding vector quantizer codebook.