Primary ciliary dyskinesia-causing mutations in Amish and Mennonite communities.

OBJECTIVE: To determine whether individuals with primary ciliary dyskinesia (PCD) from unrelated Amish and Mennonite families harbor a single and unique founder mutation. STUDY DESIGN: Subjects from Amish and Mennonite communities in several states were enrolled in the study. All subjects were clinically characterized, and nasal nitric oxide levels were measured. Nasal epithelial scrapings were collected from several subjects for ciliary ultrastructural analyses. DNA was isolated from patients with PCD and their unaffected first- and second-degree relatives. Genome-wide homozygosity mapping, linkage analyses, targeted mutation analyses, and exome sequencing were performed. RESULTS: All subjects from Old-Order Amish communities from Pennsylvania were homozygous for a nonsense mutant DNAH5 allele, c.4348C>T (p.Q1450X). Two affected siblings from an unrelated Mennonite family in Arkansas were homozygous for the same nonsense DNAH5 mutation. Children with PCD from an Amish family from Wisconsin had biallelic DNAH5 mutations, c.4348C>T (p.Q1450X) and c.10815delT (p.P3606HfsX23), and mutations in other genes associated with PCD were also identified in this community. CONCLUSION: The Amish and Mennonite subjects from geographically dispersed and socially isolated communities had the same founder DNAH5 mutation, owing to the common heritage of these populations. However, disease-causing mutations in other PCD-associated genes were also found in affected individuals in these communities, illustrating the genetic heterogeneity in this consanguineous population.

Lateral cell membranes and shunt resistance in rabbit esophageal epithelium.

BACKGROUND AND AIMS: The structures that contribute to shunt resistance (Rs) in esophageal epithelium are incompletely understood, with 35-40% of Rs known to be calcium-dependent, reflecting the role of e-cadherin. Two calcium-independent candidates for the remaining approximately 60% of Rs have been identified: the glycoprotein matrix (GPM) within stratum corneum of esophageal epithelium, and the lateral cell membranes (LCMs) from neighboring cells. METHODS: To determine the contribution of GPM and LCMs to Rs, rabbit esophageal epithelium was mounted in Ussing chambers so that transepithelial resistance (R(T)), a marker of Rs, could be monitored during luminal exposure to either glycosidases for disruption of the GPM or to hypertonic urea for separation of the LCMs. RESULTS: Glycosidases had no effect on R(T). In contrast, hypertonic urea reduced R(T), increased fluorescein flux and widened the intercellular spaces. That urea reduced R(T), and so Rs, by widening the intercellular spaces, and not by altering the e-cadherin-dependent apical junctional complex, was supported by the ability of: (a) calcium-free solution to reduce R(T) beyond that produced by urea, (b) hypertonic urea to reduce R(T) beyond that produced by calcium free solution, (c) hypertonic sucrose to collapse the intercellular spaces and raise R(T), and (d) empigen, a zwitterionic detergent, to non-osmotically widen the intercellular spaces and reduce R(T). CONCLUSION: These data indicate that the LCMs from neighboring cells are a major contributor to shunt resistance in esophageal epithelium. As resistor, they are distinguishable from the apical junctional complex by their sensitivity to (luminal) hypertonicity and insensitivity to removal of calcium.

Primary ciliary dyskinesia in Amish communities.

Primary ciliary dyskinesia is an autosomal recessive multigenic disease that results in impaired mucociliary clearance. We have diagnosed 9 subjects with primary ciliary dyskinesia from geographically dispersed Amish communities, on the basis of clinical characteristics and ciliary ultrastructural defects. Despite consanguinity, affected individuals had evidence of genetic heterogeneity.

MRT letter: Auto-fluorescence by human alveolar macrophages after in vitro exposure to air pollution particles.

Macrophages from smokers demonstrate an increased auto-fluorescence. Similarly, auto-fluorescence follows in vitro exposure of macrophages to cigarette smoke condensate (i.e., the particulate fraction of cigarette smoke). The composition of particles in cigarette smoke can be comparable to air pollution particles. We tested the postulate that macrophages exposed to air pollution particles could demonstrate auto-fluorescence. Healthy nonsmoking and healthy smoking volunteers (both 18-40 years of age) underwent fiberoptic bronchoscopy with bronchoalveolar lavage and alveolar macrophages isolated. Macrophages were incubated at 37 degrees C in 5% CO(2) with either PBS or 100 microg/mL particle for both 1 and 24 h. Particles included a residual oil fly ash, Mt. St. Helens volcanic ash, and ambient air particles collected from St. Louis, Missouri and Salt Lake City, Utah. At the end of incubation, 50 microL of the cell suspension was cytocentrifuged and examined at modes for viewing fluorescein isothiocyanate (FITC) and rhodamine fluorescence. Both emission source air pollution particles demonstrated FITC and rhodamine auto-fluorescence at 1 and 24 h, but the signal following incubation of the macrophages with oil fly ash appeared greater. Similarly, the ambient particles were associated with auto-fluorescence by the alveolar macrophages and this appeared to be dose-dependent. We conclude that exposure of macrophages to air pollution particles can be associated with auto-fluorescence in the FITC and rhodamine modes.

Ion transport and barrier function in a telomerase-immortalized human nondysplastic, Barrett's cell line (BAR-T).

Barrett's specialized columnar epithelium (SCE) replaces reflux-damaged squamous epithelium. The benefits of SCE lie in its superior protection of the esophagus against further reflux damage. It was shown that this protection is dependent on ion transport and barrier function of SCE. The risks of SCE lie in its higher predisposition to malignant transformation. An understanding of underlying mechanisms of both processes would benefit considerably from greater knowledge of the structure and function of native SCE - the latter recently advanced by the availability of a telomerase-immortalized, nonneoplastic, human Barrett's cell line (BAR-T). Some of BAR-T characteristics for growth and differentiation have been described recently, but not its capacity to serve as a model for ion transport and barrier function of SCE. To determine the latter, BAR-T cells were grown in enriched media, seeded on permeable supports, and subjected to electrical, biochemical, and morphologic study. HET-1A (esophageal epithelial cell line), a nonneoplastic, human esophageal squamous cell line, was also studied for comparison. BAR-T, but not HET-1A cells in HEPES Ringer solution behaved as polarized monolayers with the capacity for ion transport and barrier function. This was evident electrically with a volt-ohm meter (EVOM),which recorded in BAR-T a resting potential difference of 2.0 +/- 0.2 mV, Isc of 17.4 +/- 3.3 microAmps/cm2 and resistance of 103 +/- 12 ohms x cm2. Further, Isc in BAR-T was inhibitable by exposure to Na-free solution, serosal ouabain, and luminal 4-acetamido4'-isothiocyano-2,2'-stilbenedisulfonic acid. Expression of tight junction genes were determined in BAR-T and HET-1A cells using quantitative reverse transcriptase-polymerase chain reaction, with expression of zonula occludens-1 (ZO-1) set at 1 as reference. Claudins 1, 4, and 12 were prominently expressed in BAR-T (0.2-0.6 of ZO-1), while claudins 1, 11, and 12 were prominently expressed in HET-1A(0.1-0.8 of ZO-1). BAR-T, but not HET-1A, expressed claudins 4, 8, 16, 18, and 23, and HET-1A, but not BAR-T, expressed claudins 11, 15, and 20. Protein expression of prominently expressed claudins in BAR-T correlated with mRNA expression. Immunofluorescence and confocal microscopy localized claudins 1 and 4 in BAR-T to cell membranes and claudin 18, specifically to the tight junction. Membrane polarization was also documented in BAR-T by immunolocalization of NaK,ATPase to the basolateral membrane. BAR-T, but not HET-1A cells grown on permeable supports form a polarized monolayer with both ion transport and barrier function. Since a number of features of BAR-T are similar to Barrett's SCE and distinct from HET-1A, the BAR-T cell line represents a valuable resource for the study of ion transport and barrier function of nondysplastic SCE.

Palmitoylation of claudins is required for efficient tight-junction localization.

Palmitoylation of integral membrane proteins can affect intracellular trafficking, protein-protein interactions and protein stability. The goal of the present study was to determine whether claudins, transmembrane-barrier-forming proteins of the tight junction, are palmitoylated and whether this modification has functional implications for the tight-junction barrier. Claudin-14, like other members of the claudin family, contains membrane-proximal cysteines following both the second and the fourth transmembrane domains, which we speculated could be modified by S-acylation with palmitic acid. We observed that [(3)H]-palmitic acid was incorporated into claudin-14 expressed by transfection in both cultured epithelial cells and fibroblasts. Mutation of cysteines to serines following either the second or the fourth transmembrane segments decreased the incorporation of [(3)H]-palmitic acid, and mutation of all four cysteines eliminated palmitoylation. We previously reported that expression of claudin-14 in epithelial monolayers results in a fivefold increase in electrical resistance. By contrast, expression of the mutant claudin-14 resulted in smaller increases in resistance. The mutants localized less well to tight junctions and were also found in lysosomes, suggesting an alteration in trafficking or stability. However, we observed no change in protein half-life and only a small shift in fractionation out of caveolin-enriched detergent-resistant membranes. Although less well localized to the tight junction, palmitoylation-deficient claudin-14 was still concentrated at sites of cell-cell contact and was competent to assemble into freeze-fracture strands when expressed in fibroblasts. These results demonstrate that palmitoylation of claudin-14 is required for efficient localization into tight junctions but not stability or strand assembly. Decreased ability of the mutants to alter resistance is probably the result of their less efficient localization into the barrier.