Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7 Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients

https://science.sciencemag.org/content/sci/245/4922/1066.full.pdf

Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.

Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes.

Pathogenic Escherichia coli

### A. Overview

Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface receptors termed purine receptors. In this review particular emphasis is placed on the discrepancy between the

/pdf/receptors-for-purines-and-pyrimidines-56f78tnpew.pdf

Receptors for Purines and Pyrimidines

Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.

This comprehensive State of the Art review summarizes the current published knowledge base regarding the pathophysiology and microbiology of pulmonary disease in cystic fibrosis (CF). The molecular basis of CF lung disease including the impact of defective cystic fibrosis transmembrane regulator (CFTR) protein function on airway physiology, mucociliary clearance, and establishment of Pseudomonas aeruginosa infection is described. An extensive review of the microbiology of CF lung disease with particular reference to infection with P. aeruginosa is provided. Other pathogens commonly associated with CF lung disease including Staphylococcal aureus, Burkholderia cepacia, Stenotrophomonas maltophilia, Achromobacter xylosoxidans and atypical mycobacteria are also described. Clinical presentation and assessment of CF lung disease including diagnostic microbiology and other measures of pulmonary health are reviewed. Current recommendations for management of CF lung disease are provided. An extensive review of antipseudomonal therapies in the settings of treatment for early P. aeruginosa infection, maintenance for patients with chronic P. aeruginosa infection, and treatment of exacerbation in pulmonary symptoms, as well as antibiotic therapies for other CF respiratory pathogens, are included. In addition, the article discusses infection control policies, therapies to optimize airway clearance and reduce inflammation, and potential future therapies.

Pathophysiology and management of pulmonary infections in cystic fibrosis.

Cystic fibrosis transmembrane regulator (CFTR), the gene product that is mutated in cystic fibrosis (CF) patients, has a well-recognized function as a cyclic adenosine 3',5'-monophosphate (cAMP)-regulated chloride channel, but this property does not account for the abnormally high basal rate and cAMP sensitivity of sodium ion absorption in CF airway epithelia. Expression of complementary DNAs for rat epithelial Na+ channel (rENaC) alone in Madin Darby canine kidney (MDCK) epithelial cells generated large amiloride-sensitive sodium currents that were stimulated by cAMP, whereas coexpression of human CFTR with rENaC generated smaller basal sodium currents that were inhibited by cAMP. Parallel studies that measured regulation of sodium permeability in fibroblasts showed similar results. In CF airway epithelia, the absence of this second function of CFTR as a cAMP-dependent regulator likely accounts for abnormal sodium transport.

https://science.sciencemag.org/content/sci/269/5225/847.full.pdf

CFTR as a cAMP-dependent regulator of sodium channels.

The transepithelial potential difference (PD) of cystic fibrosis (CF) airway epithelium is abnormally raised and the Cl- permeability is low. We studied the contribution of active Na+ absorption to the PD and attempted to increase the Cl- permeability of CF epithelia. Nasal epithelia from CF and control subjects were mounted in Ussing chambers and were short-circuited. The basal rate of Na+ absorption was raised in CF polyps compared with control tissues. Whereas beta agonists induced Cl- secretion in normal and atopic epithelia, beta agonists further increased the rate of Na+ absorption in CF epithelia without inducing Cl- secretion. This unusual effect is not due to an abnormal CF beta receptor because similar effects were induced by forskolin, and because cAMP production was similar in normal and CF epithelia. We conclude that CF airway epithelia absorb Na+ at an accelerated rate. The abnormal response to beta agonists may reflect a primary abnormality in a cAMP-modulated path, or a normal cAMP-modulated process in a Cl- impermeable epithelial cell.

/pdf/na-transport-in-cystic-fibrosis-respiratory-epithelia-5gj6kx48c5.pdf

Na+ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation.

The epithelium of nasal tissue excised from subjects with cystic fibrosis exhibited higher voltage and lower conductance than tissue from control subjects. Basal sodium ion absorption by cystic fibrosis and normal nasal epithelia equaled the short-circuit current and was amiloride-sensitive. Amiloride induced chloride ion secretion in normal but not cystic fibrosis tissue and consequently was more effective in inhibiting the short-circuit current in cystic fibrosis epithelia. Chloride ion-free solution induced a smaller hyperpolarization of cystic fibrosis tissue. The increased voltage and amiloride efficacy in cystic fibrosis reflect absorption of sodium ions across an epithelium that is relatively impermeable to chloride ions.

https://science.sciencemag.org/content/sci/221/4615/1067.full.pdf

Abnormal ion permeation through cystic fibrosis respiratory epithelium

The effect of the number of cystic fibrosis (CF) alleles on cholera toxin (CT)-induced intestinal secretion was examined in the CF mouse model. CF mice that expressed no CF transmembrane conductance regulator (CFTR) protein did not secrete fluid in response to CT. Heterozygotes expressed 50 percent of the normal amount of CFTR protein in the intestinal epithelium and secreted 50 percent of the normal fluid and chloride ion in intestinal epithelium and secreted 50 percent of the normal fluid and chloride ion and fluid secretion suggests that CF heterozygotes might possess a selective advantage of resistance to cholera.

https://science.sciencemag.org/content/sci/266/5182/107.full.pdf

Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model

Physical stimulation of airway surfaces evokes liquid secretion, but the events that mediate this vital protective function are not understood. When cystic fibrosis transmembrane conductance regulator (CFTR) channel activity was used as a functional readout, we found signaling elements compartmentalized at both extracellular and intracellular surfaces of the apical cell membrane that activate apical Cl(-) conductance in Calu-3 cells. At the outer surface, ATP was released by physical stimuli, locally converted to adenosine, and sensed by A(2B) adenosine receptors. These receptors couple to G proteins, adenylyl cyclase, and protein kinase A, at the intracellular face of the apical membrane to activate colocalized CFTR. Thus, airways have evolved highly efficient mechanisms to "flush" noxious stimuli from airway surfaces by selective activation of apical membrane signal transduction and effector systems.

https://www.pnas.org/content/pnas/98/24/14120.full.pdf

M. J. Stutts

Papers

CFTR as a cAMP-dependent regulator of sodium channels.

Na+ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation.

Abnormal ion permeation through cystic fibrosis respiratory epithelium

Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model

Compartmentalized autocrine signaling to cystic fibrosis transmembrane conductance regulator at the apical membrane of airway epithelial cells