Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.     

Seasonal enumeration of fecal coliform bacteria from the feces of ring-billed gulls (Larus delawarensis) and Canada geese (Branta canadensis)

Water suppliers have often implicated roosting birds for fecal contamination of their surface waters. Geese and gulls have been the primary targets of this blame although literature documenting the fecal coliform content of these birds is quite limited. To determine the actual fecal coliform concentrations of these birds, fecal samples from 249 ring-billed gulls and 236 Canada geese in Westchester County, N.Y., were analyzed over a 2-year period. Results indicate that gull feces contain a greater average concentration of fecal coliform bacteria per gram (3.68 x 10(8)) than do goose feces (1.53 x 10(4)); however, average fecal sample weights of the geese were more than 15 times higher than those of the gulls.     

Therapeutic potential of the 2-alkyl and 2-alkylidene-19-nor-(20S)-modified analogs of 1alpha,25-dihydroxyvitamin D3

Five analogs of 19-nor-1alpha,25-dihydroxyvitamin D(3) are described that show highly selective and potent activities. The 2-methylene-19-nor-(20S)-1alpha25-dihydroxyvitamin D(3) (2MD) and its 2alpha-methyl sister are selectively active on the osteoblast. 2MD is bone anabolic and causes bone formation in vivo and in vitro and is being developed as a therapy for bone loss diseases such as osteoporosis. 2-Methylene-19-nor (20S)-bishomo-1alpha-hydroxypregnacalciferol (2BMP) has no activity on calcium in vivo while totally suppressing circulating parathyroid hormone. Its homologs, i.e. 2-methylene-19-nor-1alpha-hydroxy-homopregnacalciferol (2MP) and 2-methylene-19-nor-1alpha-hydroxypregnacalciferol (2MPC) act similarly but are either less selective (2MP) or not as potent (2MPC). These abbreviated side chain analogs will be developed for diseases where a rise in serum calcium is not desired, as for example, cancer, renal osteodystrophy, psoriasis and autoimmune diseases.     

Isolation and characterization of unsaturated fatty acids as natural ligands for the retinoid-X receptor

The retinoid-X receptor (RXR) is a ligand activated nuclear receptor that is the heterodimer partner for many class II nuclear receptors. Previously identified natural ligands for this receptor include 9-cis retinoic acid (9cRA), docosahexaenoic acid, and phytanic acid. Our studies were performed to determine if there are any unidentified, physiologically important RXR ligands. Agonists for RXR were purified from rat heart and testes lipid extracts with the use of a cell-based reporter assay to monitor RXR activation. Purified active fractions contained a variety of unsaturated fatty acids and components were quantified by gas-liquid chromatography of derivatized samples. The corresponding fatty acid standards elicited a similar response in the reporter cell assay. Competition binding analysis revealed that the active fatty acids compete with [3H]9cRA for binding to RXR. Non-esterified fatty acids were analyzed from lipid extracts of isolated heart and testes nuclei and endogenous concentrations were found to be within the range of their determined binding affinities. Our studies reveal tissue dependent profiles of RXR agonists and support the idea of unsaturated fatty acids as physiological ligands of RXR.     

Microstructure of anaerobic granules bioaugmented with Desulfitobacterium frappieri PCP-1

Oligonucleotide probes were used to study the structure of anaerobic granular biofilm originating from a pentachlorophenol-fed upflow anaerobic sludge bed reactor augmented with Desulfitobacterium frappieri PCP-1. Fluorescence in situ hybridization demonstrated successful colonization of anaerobic granules by strain PCP-1. Scattered microcolonies of strain PCP-1 were detected on the biofilm surface after 3 weeks of reactor operation, and a dense outer layer of strain PCP-1 was observed after 9 weeks. Hybridization with probes specific for Eubacteria and Archaea probes showed that Eubacteria predominantly colonized the outer layer, while Archaea were observed in the granule interior. Mathematical simulations showed a distribution similar to that observed experimentally when using a specific growth rate of 2.2 day(-1) and a low bacterial diffusion of 10(-7) dm(2) day(-1). Also, the simulations showed that strain PCP-1 proliferation in the outer biofilm layer provided excellent protection of the biofilm from pentachlorophenol toxicity.     

A study of the antiresorptive activity of salmon calcitonin microspheres using cultured osteoclastic cells

The purpose of this study was to evaluate salmon calcitonin (sCT) microspheres in vitro for their antiresorptive activity using cultured osteoclastic cells. The antiresorptive activity of sCT-loaded microspheres, prepared from a low molecular weight hydrophilic poly (lactide-co-glycolide) polymer (PLGA), was studied using bone marrow culture cells harvested from juvenile rats and cultured on silces of devitalized bone for up to 4 weeks. The resorptive activity of osteoclastic cells was quantified in terms of number and type of resorption pits and total area of resorption. Microspheres containing 5.1% sCT released 70% peptide in 2 weeks and 88% in 4 weeks. All sCT treatments inhibited total resorptive activity. A dose-dependent decrease in resorption was observed with sCT microspheres at 2 weeks. The high dose (10 mg of microspheres) produced a 99.5% decrease in resorption at 3 weeks, while the low dose (1 mg) produced an 80% reduction. Exposure of cultures to soluble sCT and sCT-loaded microspheres caused a decrease in the number of large pits, which were the predominant type formed in control cultures. Thus, this system could serve as an in vitro method to evaluate the antiresorptive effect of PLGA-sCT microspheres.     

Vitamin D and autoimmune diabetes

The biologically active form of vitamin D, 1,25(OH)(2)D(3), is a potent modulator of the immune system as well as a regulator of bone and mineral metabolism. Vitamin D-deficiency in infancy and vitamin D receptor gene polymorphisms may be risk factors for insulin-dependent Diabetes mellitus (IDDM). 1,25(OH)(2)D(3) and its analogs significantly repress the development of insulitis and diabetes in the non-obese diabetic (NOD) mouse, a model of human IDDM. 1,25(OH)(2)D(3) may modulate IDDM disease pathogenesis by repression of type I cytokines, inhibition of dendritic cell maturation, and upregulation of regulatory T cells. The function of vitamin D as a genetic and environmental determining factor for IDDM, the protective role of 1,25(OH)(2)D(3) and its analogs in a mouse model of IDDM, and the possible mechanisms by which this protection occurs will be reviewed.     

Formulation and in vitro transfection efficiency of poly (D, L-lactideco-glycolide) microspheres containing plasmid DNA for gene delivery

The stability, in vitro release, and in vitro cell transfection efficiency of plasmid DNA (pDNA) poly (D,L.-lactide-co-glycolide) (PLGA) microsphere formulations were investigated. PLGA microspheres containing free and polylysine (PLL)-complexed pDNA were prepared by a water-oil-water solvent extraction/evaporation technique. Encapsulation enhanced the retention of the supereoiled structure of pDNA as determined by gel electrophoresis. PLL complexation of pDNA prior to encapsulation increased both the stability of the supercoiled form and the encapsulation efficiency. Free pDNA was completely degraded after exposure to DNase while encapsulation protected the pDNA from enzymatic degradation. Rapid initial in vitro release of pDNA was obtained from microspheres containing free pDNA. while the release from microspheres containing PLL-complexed pDNA was sustained for more than 42 days. Bioactivity of encapsulated pDNA determined by in vitro cell transfection using Chinese hamster ovary cells (CHO) showed that the bioactivity of encapsulated pDNA was retained in both formulations but to a greater extent with PLL-complexed pDNA microspheres. These results demonstrated that PLGA microspheres could be used to formulate a controlledrelease delivery system for pDNA that can protect the pDNA from DNase degradation without loss of functional activity.