Differential binding of Pseudomonas aeruginosa to normal and cystic fibrosis tracheobronchial mucins

Pseudomonas aeruginosa infection is a leading cause of deteriorationof pulmonary function in patients with cystic fibrosis (CF).The interaction of the bacterium with CF and non-CF tracheobronchialmucins was examined to understand the biochemical basis forthe high susceptibility of the lungs of CF patients to infectionby P.aeruginosa. The binding of radiolabelled bacteria to puremucins in solid-phase assays was not significantly above non-specificbinding to various blocking agents, such as bovine serum albumin,Tween 20, milk powder and polyvinyl pyrrolidine. Further, therewas a tendency for the bacteria to be excluded from plasticwells and membranes coated with mucin. Therefore, an indirectapproach involving the binding of radiolabelled P.aeruginosato asialo GM₁ ganglioside, the putative receptor for the bacteriaon tracheal cells, was used to compare the interaction of CFand non-CF mucins with the bacteria. Highly purified preparationsof CF mucin were consistently better inhibitors of the bindingof the bacteria to asialo GM₁ ganglioside than non-CF mucinpreparations. In the case of the binding of a stable mucoidstrain, the difference was statistically significant (P <0.001) at all concentrations of mucin tested. For the non-mucoidstrain, the difference was significant only at the higher concentrations.Of the saccharides tested similarly, sialyl lactose and theoligosaccharide portion of asialo GM₁ were found to be goodinhibitors. The increased binding of the bacteria to CF mucinwas further confirmed by a solution binding assay in which thebinding of ¹²⁵I-labelled mucin to unlabelled bacteria was determined.The binding of the bacteria to labelled CF and non-CF mucincould be inhibited by an excess of unlabelled human tracheobronchialmucin, but not by unrelated mucins, hyaluronic acid, alginicacid, bovine serum albumin and tetramethyl urea. The higherbinding of CF mucin, particularly to the mucoid strain of P.aeruginosa,is interesting and provides a model system to further investigatethe biochemical parameters of the interaction. asialo GM₁ ganglioside cystic fibrosis Pseudomonas aeruginosa respiratory mucins saccharide inhibitors.     

Aldosterone as a renal growth factor

Aldosterone regulates blood pressure through its effects on the cardiovascular system and kidney. Aldosterone can also contribute to the development of hypertension that leads to chronic pathologies such as nephropathy and renal fibrosis. Aldosterone directly modulates renal cell proliferation and differentiation as part of normal kidney development. The stimulation of rapidly activated protein kinase cascades is one facet of how aldosterone regulates renal cell growth. These cascades may also contribute to myofibroblastic transformation and cell proliferation observed in pathological conditions of the kidney. Polycystic kidney disease is a genetic disorder that is accelerated by hypertension. EGFR-dependent proliferation of the renal epithelium is a factor in cyst development and trans-activation of EGFR is a key feature in initiating aldosterone-induced signalling cascades. Delineating the components of aldosterone-induced signalling cascades may identify novel therapeutic targets for proliferative diseases of the kidney.     

Induction of abasic sites by the drinking-water mutagen MX in Salmonella TA100

Mutagen X (MX) is a chlorinated furanone that accounts for more of the mutagenic activity of drinking water than any other disinfection by-product. It is one of the most potent base-substitution mutagens in the Salmonella (Ames) mutagenicity assay, producing primarily GC to TA mutations in TA100. MX does not produce stable DNA adducts in cellular or acellular DNA. However, theoretical calculations predict that it might induce abasic sites, which it does in supercoiled plasmid DNA but not in rodents. To investigate the ability of MX to induce abasic sites in cellular DNA, we used an aldehydic site assay to detect abasic sites in DNA from Salmonella TA100 cells treated for 1.5 h with MX. At 0, 2.3, and 4.6 μM, MX induced mutant frequencies (revertants/10⁶ survivors) and percent survivals of 2 (100%), 14.9 (111%), and 59.3 (45%), respectively. The frequencies of abasic sites (sites/10⁵ nucleotides) for the control and two concentrations were 5.9, 6.2, and 9.7, respectively, with the frequency at the highest concentration being significant (P < 0.001). These results provide some evidence for the ability of MX to induce abasic sites in cellular DNA. However, the lack of a dose response makes it unclear whether this DNA damage underlies the mutagenic activity of MX.     

Comparison of physical and genetic properties of palindromic DNA sequences.

Some viable palindromic DNA sequences were found to cause an increase in the recovery of genetic recombinants. Although these palindromes contained no Chi sites, their presence in cis caused apparent recA+-dependent recombination to increase severalfold. This biological property did not correlate with the physical properties of the palindromes' extrusion of cruciform structures in vitro. Thus, two unrelated palindromes with similar effects on recombination in both Escherichia coli and Pseudomonas syringae displayed quite different kinetics of cruciform formation. In plasmids of native superhelical density, one palindrome underwent rapid cruciform formation at 55 degrees C, whereas the other did not form detectable cruciforms at any temperature. A shorter palindrome with similarly rapid kinetics of cruciform formation did not affect recombination detectably. The lack of a clear relationship between physical and genetic properties was also demonstrated in the case of longer, inviable palindromes. Here we found that the degree of asymmetry required in vivo to rescue a long palindrome from inviability far exceeded that required to kinetically prohibit cruciform extrusion in vitro.     

Metabolic cooperation between vascular endothelial cells and smooth muscle cells in co-culture: changes in low density lipoprotein metabolism

A microcarrier co-culture system for aortic endothelial cells and smooth muscle cells (SMCs) was developed as a model for metabolic interactions between cells of the vessel wall. Low density lipoprotein (LDL) metabolism in SMCs was significantly influenced by co-culture with endothelium. The numbers of high affinity receptors for LDL was increased more than twofold (range, 2.1-5.6), with concomitant increases in LDL receptor-mediated endocytosis and degradation. These effects reached a plateau at an endothelial cell/SMC ratio of 1. Kinetic analysis of the endocytic pathway for LDL in SMCs indicated that, in co-culture with endothelium, there was no alteration in the binding affinity of LDL to its receptors but that the internalization rate constant declined and the rate constant for degradation increased. This analysis suggested that the formation and migration of endocytic vesicles was the rate-limiting step of enhanced LDL metabolism under co-culture conditions. Two mechanisms by which endothelial cells influenced smooth muscle LDL metabolism were identified. First, mitogen(s) derived from endothelial cells stimulated entry of SMCs into the growth cycle, and the changes in LDL metabolism occurred as a consequence of G1-S transition. Second, SMC lipoprotein metabolism was stimulated in the absence of mitogens by a low molecular weight (less than 3,500) factor or factors. Co-culture was a required condition for the latter effect, suggesting that the mediator(s) may be unstable or that cell-cell communication was necessary for expression. These results (a) demonstrate that vascular cell interactions can modify LDL metabolism in SMCs, (b) provide some insights into the mechanisms responsible, and (c) identify co-culture as an experimental approach appropriate to certain aspects of vascular cell biology.