Twenty-five years of selection for improved leg health in purebred broiler lines and underlying genetic parameters.

Leg health is an important component of broiler welfare and the economics of broiler production. This study presents the development of leg health in 3 purebred commercial broiler lines during 25 yr of selection and investigates the genetic background of leg health traits in current populations of these lines. The leg health traits were deformities of the long bones (LD) and crooked toes (CT), recorded since 1985, and tibial dyschondroplasia (TD) and hock burn (HB), recorded since 1990. The prevalence of CT and HB decreased mainly in the first decade (range among lines -1.2 to -2.3% and -1.3 to -1.5% per year, respectively), after which it stabilized at low levels. The prevalence of LD and TD decreased by -0.6 to -0.9% and -0.4 to -1.2% per year, respectively. Genetic parameters were estimated using data from 4 recent generations. The BW ranged from 2.0 to 2.4 kg at 5 wk of age; the prevalences of LD, CT, TD, and HB from 8.6 to 12.9%, 0.6 to 2.6%, 4.6 to 8.0%, and 4.0 to 12.2%, respectively. Estimates of heritability were 0.04 to 0.07 for LD, 0.01 to 0.10 for CT, 0.10 to 0.27 for TD, and 0.06 to 0.09 for HB (all SE ≤0.01). Estimates of the genetic correlations between LD and CT were 0.11 to 0.43 (all SE ≤0.09), between these traits and HB were negligible, and of TD with LD, CT, and HB were -0.26 to 0.16 (all SE ≤0.11). Estimates of genetic correlations between the leg health traits and BW were lowly to moderately unfavorable, ranging from 0.09 to 0.37 (all SE ≤0.06). The differences between the lines suggest that strategies for simultaneous improvement of all traits tailored for each line individually have been effective. This research demonstrates the long-term effectiveness of selection for improving leg health in broilers and highlights that, despite somewhat unfavorable genetic correlations with BW, these traits can be improved simultaneously in a balanced breeding program.

Evidence against the acidification hypothesis in cystic fibrosis.

The pleiotropic effects of cystic fibrosis (CF) result from the mislocalization or inactivity of an apical membrane chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR may also modulate intracellular chloride conductances and thus affect organelle pH. To test the role of CFTR in organelle pH regulation, we developed a model system to selectively perturb the pH of a subset of acidified compartments in polarized cells and determined the effects on various protein trafficking steps. We then tested whether these effects were observed in cells lacking wild-type CFTR and whether reintroduction of CFTR affected trafficking in these cells. Our model system involves adenovirus-mediated expression of the influenza virus M2 protein, an acid-activated ion channel. M2 expression selectively slows traffic through the trans-Golgi network (TGN) and apical endocytic compartments in polarized Madin-Darby canine kidney (MDCK) cells. Expression of M2 or treatment with other pH perturbants also slowed protein traffic in the CF cell line CFPAC, suggesting that the TGN in this cell line is normally acidified. Expression of functional CFTR had no effect on traffic and failed to rescue the effect of M2. Our results argue against a role for CFTR in the regulation of organelle pH and protein trafficking in epithelial cells.

Reconstituting the barrier properties of a water-tight epithelial membrane by design of leaflet-specific liposomes.

To define aspects of lipid composition and bilayer asymmetry critical to barrier function, we examined the permeabilities of liposomes that model individual leaflets of the apical membrane of a barrier epithelium, Madin-Darby canine kidney type 1 cells. Using published lipid compositions we prepared exofacial liposomes containing phosphatidylcholine, sphingomyelin, glycosphingolipids, and cholesterol; and cytoplasmic liposomes containing phosphatidylethanolamine, phosphatidylserine, and cholesterol. The osmotic permeability of cytoplasmic liposomes to water (P(f)), solutes, and NH(3) was 18-90-fold higher than for the exofacial liposomes (P(f(ex)) = 2.4 +/- 0.4 x 10(-4) cm/s, P(f(cy)) = 4.4 +/- 0.3 x 10(-3) cm/s; P(glycerol(ex)) = 2.5 +/- 0.3 x 10(-8) cm/s, P(glycerol(cy)) = 2.2 +/- 0.02 x 10(-6) cm/s; P(NH3(ex)) = 0. 13 +/- 0.4 x 10(-4) cm/s, P(NH3(cy)) = 7.9 +/- 1.0 x 10(-3) cm/s). By contrast, the apparent proton permeability of exofacial liposomes was 4-fold higher than cytoplasmic liposomes (P(H+(ex)) = 1.1 +/- 0. 1 x 10(-2) cm/s, P(H+(cy)) = 2.7 +/- 0.6 x 10(-3) cm/s). By adding single leaflet permeabilities, we calculated a theoretical P(f) for a Madin-Darby canine kidney apical membrane of 4.6 x 10(-4) cm/s, which compares favorably with experimentally determined values. In exofacial liposomes lacking glycosphingolipids or sphingomyelin, permeabilities were 2-7-fold higher, indicating that both species play a role in barrier function. Removal of cholesterol resulted in 40-280-fold increases in permeability. We conclude: 1) that we have reconstituted the biophysical properties of a barrier membrane, 2) that the barrier resides in the exofacial leaflet, 3) that both sphingomyelin and glycosphingolipids play a role in reducing membrane permeability but that there is an absolute requirement for cholesterol to mediate this effect, 4) that these results further validate the hypothesis that each leaflet offers an independent resistance to permeation, and 5) that proton permeation was enhanced by sphingolipid/cholesterol interactions.

Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells.

Channel gating of the cystic fibrosis transmembrane conductance regulator (CFTR) is activated in response to cAMP stimulation. In addition, CFTR activation may also involve rapid insertion of a subapical pool of CFTR into the plasma membrane (PM). However, this issue has been controversial, in part because of the difficulty in distinguishing cell surface vs. intracellular CFTR. Recently, a fully functional, epitope-tagged form of CFTR (M2-901/CFTR) that can be detected immunologically in nonpermeabilized cells was characterized (Howard M, Duvall MD, Devor DC, Dong J-Y, Henze K, and Frizzell RA. Am J Physiol Cell Physiol 269: C1565-C1576, 1995; and Schultz BD, Takahashi A, Liu C, Frizzell RA, and Howard M. Am J Physiol Cell Physiol 273: C2080-C2089, 1997). We have developed replication-defective recombinant adenoviruses that express M2-901/CFTR and used them to probe cell surface CFTR in forskolin (FSK)-stimulated polarized Madin-Darby canine kidney (MDCK) cells. Virally expressed M2-901/CFTR was functional and was readily detected on the apical surface of FSK-stimulated polarized MDCK cells. Interestingly, at low multiplicity of infection, we observed FSK-stimulated insertion of M2901/CFTR into the apical PM, whereas at higher M2-901/CFTR expression levels, no increase in surface expression was detected using indirect immunofluorescence. Immunoelectron microscopy of unstimulated and FSK-stimulated cells confirmed the M2-901/CFTR redistribution to the PM upon FSK stimulation and demonstrates that the apically inserted M2-901/CFTR originates from a population of subapical vesicles. Our observations may reconcile previous conflicting reports regarding the effect of cAMP stimulation on CFTR trafficking.

Enhanced channelling of sulphate through a rapidly exchangeable sulphate pool in response to stimulated glycosaminoglycan synthesis in pancreatic epithelial cells.

The ability of cells to decorate glycosaminoglycans (GAGs) with sulphate in highly specific patterns is important to extracellular matrix biogenesis and placing appropriate glycosulphated ligands on the cell surface. We have examined sulphate metabolism in two pancreatic duct epithelial cell lines - PANC-1 and CFPAC-1 (derived from a cystic fibrosis patient) with a view to understanding how pancreatic cells utilise intracellular sulphate. [35S]Sulphate uptake was rapid and reached near steady state levels within 10 min. However, the intracellular specific activity of [35S]sulphate for PANC-1 and CFPAC-1 reached only 35 and 10%, respectively, of the medium specific activity at 10 min. Therefore, sulphate appears to reside within two compartments; a rapidly exchangeable sulphate pool (RESP) and a slowly exchangeable sulphate pool (SESP). Reducing chloride in the medium, increased the specific activity of [35S]sulphate within cells and increased the size of the inorganic sulphate pool, suggesting that the RESP was enlarged. Sulphate pools were not different in size between the two cell lines in physiological NaCl. Increasing the size of the sulphate pool had no effect on [35S]sulphate:[3H]glucosamine ratios incorporated into glycosaminoglycans (GAGs); however, stimulating the synthesis of GAGs with 4-methylumbelliferyl-beta-d-xyloside, stably elevated [35S]:[3H] ratios. This was due to higher [35S]sulphate incorporation. [35S]Cysteine contributed less than 0.1% of the cells' sulphate requirements. We conclude that in the face of elevated demand for sulphate, pancreatic cells appear to channel a greater proportion through the RESP.

Glycosylation differences between a cystic fibrosis and rescued airway cell line are not CFTR dependent.

Altered glycosylation of mucus and membrane glycoconjugates could explain reported differences in binding of bacterial pathogens to cystic fibrosis (CF) versus normal tissue. However, because bacteria can alter cell surface glycoconjugates, it is not possible to assess the role of cystic fibrosis transmembrane conductance regulators (CFTR) in glycosylation in these studies. To address this issue, we have developed quantitative lectin binding assays to compare cell surface glycosylation in well-matched immortalized CF cells and rescued cell lines. The CF airway bronchial epithelial cell line IB3-1 consistently bound more peanut agglutinin (PNA) than its clonal derivative S9, which stably expresses functional wild-type CFTR. Pretreatment with neuraminidase increased PNA binding and abolished the difference between the two cell lines. However, infection of the IB3-1 cells with a replication-deficient recombinant adenovirus encoding CFTR restored CFTR function but did not alter PNA binding to cells. In contrast, treatment with the weak base ammonium chloride increased PNA binding to both cell lines as expected. Our data show that even clonally related CF and rescued cells can exhibit significant differences in carbohydrate processing. Although the differences that we found are consistent with the proposed role for CFTR in modulating intraorganellar pH, our data strongly suggest that they are CFTR independent. These studies add a cautionary note to the interpretation of differences in glycosylation between CF and normal primary tissues and immortalized cells.

Sulfation of chondroitin/dermatan sulfate by cystic fibrosis pancreatic duct cells is not different from control cells.

Cystic fibrosis is associated with mutations of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated plasma membrane chloride channel. Cystic fibrosis patients have been reported to possess elevated sulfation of glycoconjugates, which may contribute to the pathogenesis of the disease. Sulfation of glycosaminoglycans by a cystic fibrosis pancreatic adenocarcinoma cell line homozygous for DeltaF508 (CFPAC-1), a control pancreatic cell line (PANC-1), two CFPAC-1 cell lines transfected with the gene for CFTR (PLJ-CFTR-4.7, TR20), and a mock-transfected CFPAC-1 control (PLJ-6) was investigated. Cells were radiolabeled with [35S]sulfate and [3H]glucosamine, and glycosaminoglycans secreted into the medium after 24 and 72 h were isolated. Chondroitinase ABC digestion of chondroitin/dermatan sulfate allowed the recovery of disaccharides which were analyzed for their degree of sulfation by strong anion-exchange HPLC. No differences in the extent of sulfation by any of the cell lines were noted. However, glycoaminoglycans synthesized by cystic fibrosis cells consistently exhibited twofold higher [35S]-sulfate:[3H]glucosamine ratios than the controls. We conclude that CFTR plays no role in the sulfation of chondroitin/dermatan sulfate by pancreatic cells and that isotope incorporation ratios alone are insufficient evidence of changes in sulfation levels.

Organ-specific over-sulfation of glycosaminoglycans and altered extracellular matrix in a mouse model of cystic fibrosis.

Cystic fibrosis (CF) is a fatal inherited disease caused by the loss of function of a plasma membrane chloride channel-the cystic fibrosis transmembrane conductance regulator (CFTR). It is characterized by viscous mucous secretions which have abnormal glycosylation and sulfation. The development of a CFTR knockout mouse has allowed in vivo experiments aimed at investigating the over-sulfation phenomenon reported for CF glycoconjugates. Four CF and five control mice injected with [35S]sulfate were examined for differences in the sulfation of glycosaminoglycans (GAGs) synthesized by 12 tissues after 48 h. The liver and pancreas of CF mice incorporated significantly higher amounts of [35S]sulfate into GAGs (dpm/microg) than the controls, while the ileum, jejunum, colon, cecum, spleen, trachea, and gall bladder of CF mice exhibited higher incorporation levels that were not significant. The lung and nasal septum were not different, and the nasal mucosa of CF mice was significantly lower (P < 0.05). Structural analysis of the chondroitin/dermatan sulfate component by strong anion-exchange HPLC revealed that the liver and ileum of CF mice incorporated significantly more total sulfate than controls. However, for other organs, the explanation for higher isotope incorporation was a 40-50% higher specific activity of [35S]sulfate within GAGs. This finding implied different uptake kinetics of sulfate from the circulation or that CF mice have altered sulfate pools. CF mice also had altered proportions of chondroitin/dermatan sulfate to heparan sulfate in the ileum and gall bladder (P < 0.05). We conclude that extracellular matrix architecture in some CF organs may be abnormal and that sulfation of glycoconjugates by some organs and sulfate utilization in others have been affected by the loss of CFTR. This study provides the first in vivo evidence for an influence of CFTR on glycoconjugate sulfation and suggests other secondary manifestations of CFTR dysfunction associated with abnormalities of the extracellular matrix.

Likelihood methods for locating disease genes in nonequilibrium populations.

Until recently, attempts to map disease genes on the basis of population associations with linked markers have been based on expected values of linkage disequilibrium. These methods suffer from the large variances imposed on disequilibrium measures by the evolutionary process, but a more serious problem for many diseases is that they assume an equilibrium population. For diseases that arose only a few hundred generations ago, it is more appropriate to concentrate on the initial growth phase of the disease. We invoke a Poisson branching process for this early growth, and estimate the likelihood for the recombination fraction between marker and disease loci, on the basis of simulated disease populations. The limits of the resulting support intervals for the recombination fraction vary inversely with the age of the disease in generations. We illustrate the procedure with data on cystic fibrosis and diastrophic dysplasia, for which the method appears appropriate, and for Friedreich ataxia and Huntington disease, for which it does not. A valuable aspect of the method is the ability in some cases to compare likelihoods of the three orders for a disease locus and two linked marker loci.

Effects of multiple retrovirus insertions on quantitative traits of mice.

To assess the potential to generate quantitative genetic variation by insertional mutagenesis in a vertebrate, lines of mice in which many provirus vector inserts segregated at a low initial frequency on an inbred background (insert lines) were subjected to divergent artificial selection on body weight at 6 weeks and responses and heritability estimates compared to control lines lacking inserts. Heritability estimates were more than 1.5 times greater in the insert lines than in the controls, but because the phenotypic variance was substantially higher in the insert lines the genetic variance was about 3 times greater. Realized heritability estimates tended to be lower than heritabilities estimated by an animal model which utilizes information in covariances between all relatives in the data set. A surprisingly large response to selection occurred in the inbred control line. Insert lines were about 20% less fertile than controls. Division of the selection lines into inbred sublines in the later generations of the experiment revealed substantially greater variation among sublines of the insert lines than among the controls. Heritabilities were similar to typical estimates for the trait in outbred populations. In conclusion, there was clear evidence of extra variation deriving from inserts, which has yet to be attributed to individual genes.