Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that affect protein structure and channel function. CFTR, localized in the apical membrane within cholesterol and sphingomyelin rich regions, is an ABC transporter that functions as a chloride channel. Here, we report that expression of defective CFTR (#F508CFTR or decreased CFTR) in human lung epithelial cell lines increases sphingolipid synthesis and mass of sphinganine, sphingosine, four long-chain saturated ceramide species, C16 dihydroceramide, C22, C24, C26-ceramide, and sphingomyelin, and decreases mass of C18 and unsaturated C18:1 ceramide species. Decreased expression of CFTR is associated with increased expression of longchain base subunit 1 of serine-palmitoyl CoA, the rate-limiting enzyme of de novo sphingolipid synthesis and increased sphingolipid synthesis. Overexpression of #F508CFTR in bronchoalveolar cells that do not express CFTR increases sphingolipid synthesis and mass, whereas overexpression of wild-type CFTR, but not of an unrelated ABC transporter, ABCA7, decreases sphingolipid synthesis and mass. The data are consistent with a model in which CFTR functions within a feedback system that affects sphingolipid synthesis and in which increased sphingolipid synthesis could reflect a physiological response to sequestration of sphingolipids or altered membrane structure.-Hamai, H., F. Keyserman, L. M. Quittell, and T. S. Worgall. Defective CFTR increases synthesis and mass of sphingolipids that modulate membrane composition and lipid signaling.

Small dense low density lipoprotein (LDL) particles have altered apolipoprotein (apo) B conformation and lowered affinity for the LDL receptor ( J. Biol. Chem. 1994. 269: 511-519). Herein, we examine the interaction of small dense LDL with cell LDL receptor-independent binding sites. Compared to normal LDL, at low LDL cell media concentrations ( Ͻ 10 g/ml), small dense LDL had decreased specific binding to the LDL receptor on normal fibroblasts at 4 Њ C, but a 2-fold increased binding to LDL receptor-independent cell sites. At higher LDL concentration (100 g/ ml), LDL receptor-independent binding of small dense LDL was 4.5-fold that of normal LDL in normal fibroblasts, but greater (2-to 14-fold) in LDL receptor-negative fibroblasts. In LDL receptor-negative fibroblasts at 37 ؇ C, small dense LDL had higher (3-fold) cell association than normal size LDL but no effective LDL degradation. At high LDL concentrations ( Ն 100 g/ml), LDL binding to normal or LDL receptor-negative fibroblasts was not affected by several anti-apoB monoclonal antibodies or by cell pretreatment with proteases, chondroitinase, or neuraminidase. In contrast, pretreating normal and receptor-negative fibroblasts with heparinase and heparitinase decreased LDL cell binding by 35% and 50%, respectively. Similarly, preincubation of receptor-negative fibroblasts with sodium chlorate, an inhibitor of proteoglycan sulfation, decreased LDL binding by about 45%. We hypothesize that small dense LDL might be more atherogenic than normal size LDL due to decreased hepatic clearance by the LDL receptor, and enhanced anchoring to LDL receptor-independent binding sites in extrahepatic tissues (e.g., the arterial wall), a process mediated, in part, by cell surface proteoglycans.-Galeano, N. F., M. Al-Haideri, F. Keyserman, S. C. Rumsey, and R. J. Deckelbaum. Small dense low density lipoprotein has increased affinity for LDL receptor-independent cell surface binding sites: a potential mechanism for increased atherogenicity.

The fragments responsible for the immunodiffusion reactivity of middle- and low-density fractions of trypsin-digested bovine nasal cartilage proteoglycan have been identified and obtained in relatively homogeneous fractions. Glycosaminoglycan-bearing tryptic fragments were isolated from 4 M guanidinium chloride extracts of cartilage by ion-exchange chromatography and fractionated by dissociative equilibrium density gradient ultracentrifugation at a starting density of 1.50. Fragments in the middle fractions of the density gradient were digested with chondroitinase ABC and subfractionated by Sepharose 6B column chromatography. Middle-density subfractions contained fragments which were chemically and immunologically identical to those in high-density fragment subfractions of similar elution from Sepharose 6B. The middle-density subfractions contained two additional immunoprecipitating fragments. One, with alanine as N-terminal amino acid, was isolated by virtue of its retention by a column of concanavalin A-Sepharose 4B and its resistance to digestion with keratanase; the second was concentrated in a subfraction whose elution from concanavalin A-Sepharose 4B was retarded. The gradient fraction of lowest density contained fragments with the properties of the major tryptic fragments of the hyaluronic acid-binding segment of the proteoglycan monomer and the link proteins. These were recovered as a complex in the void volume upon Sepharose gel chromatography in saline-buffer and were resolved into relatively homogeneous fractions by column chromotography on CL-Sepharose 6B in 4 M guanidinium chloride. In all, tryptic digests of cartilage proteoglycan contain at least seven different immunoprecipitating fragments, some of which may not have been correctly identified previously.