Recently, we demonstrated a net blood-to-brain passage of the oxysterol 27-hydroxycholesterol corresponding to 4-5 mg/day. As the steady-state levels of this sterol are only 1-2 mg/g brain tissue, we hypothesized that it is metabolized and subsequently eliminated from the brain. To explore this concept, we first measured the capacity of in vitro systems representing the major cell populations found in the brain to metabolize 27-hydroxycholesterol. We show here that 27-hydroxycholesterol is metabolized into the known C 27 steroidal acid 7a-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7a-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7a-hydroxy-3-oxo-4-cholestenoic acid in plasma from the internal jugular vein and brachial artery of healthy volunteers. Calculation of the arteriovenous concentration difference revealed a significant in vivo flux of this steroid from the brain into the circulation in human.Together, these studies identify a novel metabolic route for the elimination of 27-hydroxylated sterols from the brain. Given the emerging connections between cholesterol and neurodegeneration, this pathway may be of importance for the development of these conditions.

Pulse-chase experiments in the colon cell line LS 174T combined with subcellular fractionation by sucrose density gradient centrifugation showed that the initial dimerization of the MUC2 apomucin started directly after translocation of the apomucin into the rough endoplasmic reticulum as detected by calnexin reactivity. As the mono-and dimers were chased, O-glycosylated MUC2 mono-and dimers were precipitated using an O-glycosylation-insensitive antiserum against the N-terminal domain of the MUC2 mucin. These O-glycosylated species were precipitated from the fractions that comigrated with the galactosyltransferase activity during the subcellular fractionation, indicating that not only MUC2 dimers but also a significant amount of monomers are transferred into the Golgi apparatus. Inhibition of N-glycosylation with tunicamycin treatment slowed down the rate of dimerization and introduced further oligomerization of the MUC2 apomucin in the endoplasmic reticulum. Results of two-dimensional gel electrophoresis demonstrated that these oligomers (putative tri-and tetramers) were stabilized by disulfide bonds. The non-N-glycosylated species of the MUC2 mucin were retained in the endoplasmic reticulum because no Oglycosylated species were precipitated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MUC2 mucin.The mucus layer on the epithelial surface of the mucous membrane is mainly made up of water and the gel-forming components, the mucus glycoproteins, or mucins, consisting of more than 50% O-linked oligosaccharides (1, 2). The peptide chain of mucins has domains with a high abundance of Ser, Thr, and Pro, usually in repetitive sequences (tandem repeats). The oligosaccharide chains are O-linked to Ser and Thr, thereby forming highly glycosylated domains or mucin domains.The apoprotein of the human intestinal MUC2 mucin, which is fully sequenced, contains two mucin domains with large amounts of the amino acids Thr, Pro, and Ser (3, 4); the larger of these domains consists of well conserved 23-amino acid repeated sequences. The mucin domains are flanked by Cys-rich domains; one C-terminal, one N-terminal, and one central domain. The carboxyl and amino termini of the human MUC2 mucin and the blood coagulation factor, the von Willebrand factor (vWF), 1 show sequence similarities in the positions of the cysteines. The vWF forms disulfide-bonded dimers between two C termini, and the N termini mediate further oligomerization (5). We have earlier shown that the human MUC2 apomucin forms dimers before being O-glycosylated (6). To study the initial assembly of the human MUC2 mucin in more detail, pulse-chase labeling and subcellular fractionation has been performed on LS 174T cells. An early dimerization was observed in the endoplasmic reticulum; there was no further oligomerization, and the dimerization was followed by O-glycosylation of the mono-and dimer in the Golgi apparatus. Tunicamycin treatment slowed down the dimerization rate, introduce...

Addition of the weak base ammonium chloride (NH4Cl) or the proton pump inhibitor bafilomycin A1 to cultured HeLa and LS 174T cells effectively neutralized the pH gradient of the secretory pathway. This resulted in relocalization of the three studied glycosyltransferases, N-acetylgalactosaminyltransferase 2, beta1,2 N-acetylglucosaminyltransferase I, and beta1,4 galactosyltransferase 1, normally localized to the Golgi stack, the medial/trans-Golgi and the trans-Golgi/TGN, respectively. Indirect immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation of the tagged or native glycosyltransferases showed that NH4Cl caused a relocalization of the enzymes mainly to vesicles of endosomal type, whereas bafilomycin A1 gave mainly cell surface staining. The general morphology of the endoplasmic reticulum and Golgi apparatus was retained as judged from immunofluorescence and electron microscopy studies. When the O-glycans on the guanidinium chloride insoluble gel-forming mucins from the LS 174T cells were analyzed by gas chromatography-mass spectrometry after neutralization of the secretory pathway pH by NH4Cl over 10 days shorter O-glycans were observed. However, no decrease in the number of oligosaccharide chains was indicated. Together, the results suggest that pH is a contributing factor for proper steady-state distribution of glycosyltransferases over the Golgi apparatus and that altered pH may cause alterations in glycosylation possibly due to a relocalization of glycosyltransferases.

Early in mitosis, the mammalian Golgi apparatus disassembles, and fluorescence microscopy reveals Golgi clusters and an extensive, nonresolvable haze that either represents scattered vesicles or a merged endoplasmic reticulum (ER)-Golgi compartment. To help decide between these alternatives, we have carried out a combined microscopic and pharmacological analysis, by using a BS-C-1 cell line stably coexpressing ER and Golgi markers. Video fluorescence microscopy showed that these two organelles were morphologically distinguishable at all stages of mitosis, and photobleaching experiments showed that diffusion of the Golgi marker was unaffected by the presence of the ER. Fragmentation of the ER by using filipin III completely blocked diffusion of the ER marker but had no effect on the Golgi marker, unless it was first relocated to the ER by using brefeldin A. The Golgi haze was also studied using BODIPY ceramide. Its diffusion was slower in mitotic Golgi than in mitotic ER, but similar to that of a Golgi enzyme marker in the mitotic Golgi haze or in Golgi vesicles generated by ilimaquinone. Together, these results support the idea that the Golgi and the ER remain separate during mitosis and strongly suggest that Golgi markers move by vesicle diffusion, as opposed to lateral diffusion in continuous membranes. INTRODUCTIONThe status of the Golgi apparatus as an organelle has been the subject of a long-running debate that focused initially on the route taken by transiting cargo (cisternal maturation or vesicle-mediated transport; Glick and Malhotra, 1998;Pelham and Rothman, 2000) and, more recently, on the mechanisms of duplication and partitioning that underlie its biogenesis (Marsh and Howell, 2002;Munro, 2002). If the Golgi apparatus is responsible for its biogenesis, then it can be viewed as an autonomous organelle (Pelletier et al., 2002). If, on the other hand, it depends on the endoplasmic reticulum, then it can be viewed as a dependent organelle, which exists only as a consequence of ER functioning (Zaal et al., 1999;Bevis et al., 2002;Munro, 2002).Golgi partitioning during mitosis in animal cells has been particularly controversial, with models ranging from partitioning by Golgi elements themselves (Lucocq et al., 1989;Misteli and Warren, 1995;Jesch and Linstedt, 1998;Shima et al., 1998;Jesch et al., 2001;Jokitalo et al., 2001) to the partial or even complete merger of the Golgi with the ER, which then mediates the partitioning process (Thyberg and Moskalewski, 1992;Zaal et al., 1999;Kano et al., 2000;Terasaki, 2000). Although most biochemical experiments have yielded consistent results, suggesting a separation of the ER and Golgi during mitosis (Jesch and Linstedt, 1998;Farmaki et al., 1999;Jesch et al., 2001), microscopic experiments have often yielded contradictory results (Thyberg and Moskalewski, 1992;Shima et al., 1998;Zaal et al., 1999;Jokitalo et al., 2001).A particular issue has been the Golgi haze observed by fluorescence microscopy of mitotic cells. Breakdown and fragmentation of the Golgi ribbon a...