Here we report a new isolation method for mouse glomeruli. The method is fast and simple and allows for the isolation of virtually all glomeruli present in the adult mouse kidney with minimal contamination of nonglomerular cells. Mice were perfused through the heart with magnetic 4.5-m diameter Dynabeads. Kidneys were minced into small pieces, digested by collagenase, filtered, and collected using a magnet. The number of glomeruli retrieved from one adult mouse was 20,131 ؎ 4699 (mean ؎ SD, n ‫؍‬ 14) with a purity of 97.5 ؎ 1.7%. The isolated glomeruli retained intact morphology, as confirmed by light and electron microscopy, as well as intact mRNA integrity, as confirmed by Northern blot analysis. The method was applicable also to newborn mice, which allows for the isolation of immature developmental stage glomeruli. This method makes feasible transcript profiling and proteomic analysis of the developing, healthy and diseased mouse glomerulus. As the genome projects are near completion, 1,2 an important step in the functional analysis of genome data are the determination of transcriptomes corresponding to specific cellular functions and states of differentiation. Such analyses require methods allowing for the isolation of highly homogenous population of cells and/or microorgans from in vivo situations. One such microorgan is the kidney glomerulus. Glomeruli constitute ϳ10% of whole kidney tissues and are unique structures of microvasculature mainly made up of three highly specialized cell types; fenestrated endothelial cells, mesangial cells, and podocytes. These cell types together with the glomerular basement membrane form the permeable barrier across which blood is filtered to produce primary urine. During the past decade several gene products have been shown to play essential roles in glomerulus development, 3-5 function, and pathology. 6 However, our knowledge of the molecular mechanisms governing glomerulus morphogenesis and development of the specialized features of its individual cells is still very limited. An obvious difficulty in addressing these issues stems from the low abundance of the glomerulus cells and the inability of the glomerulus cell types to retain their differentiated features in cell culture. Podocytes, for example, make up less than 2% of kidney tissues. Although endothelial cells and pericytes exist outside the glomerulus, their phenotype within the glomerulus is quite distinct from related cells elsewhere. 7 We describe a new protocol for the isolation of glomeruli from mice. The protocol is fast and allows for the isolation of virtually all glomeruli present in a mouse kidney at 97% purity. The method thus allows for transcript profiling and proteomic analysis of the glomerulus using standard procedures.

Loss of pericytes from the capillary wall is a hallmark of diabetic retinopathy, however, the pathogenic signi®cance of this phenomenon is unclear. In previous mouse gene knockout models leading to pericyte de®-ciency, prenatal lethality has so far precluded analysis of postnatal consequences in the retina. We now report that endothelium-restricted ablation of platelet-derived growth factor-B generates viable mice with extensive inter-and intra-individual variation in the density of pericytes throughout the CNS. We found a strong inverse correlation between pericyte density and the formation of a range of retinal microvascular abnormalities strongly reminiscent of those seen in diabetic humans. Proliferative retinopathy invariably developed when pericyte density was <50% of normal. Our data suggest that a reduction of the pericyte density is suf®cient to cause retinopathy in mice, implying that pericyte loss may also be a causal pathogenic event in human diabetic retinopathy.

Normal blood microvessels are lined by pericytes, which contribute to microvessel development and stability through mechanisms that are poorly understood. Pericyte deficiency has been implicated in the pathogenesis of microvascular abnormalities associated with diabetes and tumors. However, the unambiguous identification of pericytes is still a problem because of cellular heterogeneity and few available molecular markers. Here we describe an approach to identify pericyte markers based on transcription profiling of pericyte-deficient brain microvessels isolated from platelet-derived growth factor (PDGF-B)-/- and PDGF beta receptor (PDGFRbeta)-/- mouse mutants. The approach was validated by the identification of known pericyte markers among the most down-regulated genes in PDGF-B-/- and PDGFRbeta-/- microvessels. Of candidates for novel pericyte markers, we selected ATP-sensitive potassium-channel Kir6.1 (also known as Kcnj8) and sulfonylurea receptor 2, (SUR2, also known as Abcc9), both part of the same channel complex, as well as delta homologue 1 (DLK1) for in situ hybridization, which demonstrated their specific expression in brain pericytes of mouse embryos. We also show that Kir6.1 is highly expressed in pericytes in brain but undetectable in pericytes in skin and heart. The three new brain pericyte markers are signaling molecules implicated in ion transport and intercellular signaling, potentially opening new windows on pericyte function in brain microvessels.

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...

The MUC2 mucin is the major gel-forming mucin in the small and large intestine. Due to its sequence similarities with the von Willebrand factor, it has been suggested to dimerize in the endoplasmic reticulum and polymerize in the trans-Golgi network. Using an O-glycosylation-sensitive MUC2 antiserum, a dimerization has been shown to occur in the endoplasmic reticulum of LS 174T cells (Asker, Chem. 268, 18771-18781). Reduction of the mucins followed by purification by isopycnic density gradient ultracentrifugation and analysis by agarose gel electrophoresis revealed two bands reacting with an anti-MUC2 tandem repeat antibody after deglycosylation. These bands migrated identically to the bands shown by metabolic labeling, and they could also be separated by rate zonal ultracentrifugation. These results suggest that the MUC2 mucin is forming nonreducible intermolecular bonds early in biosynthesis, but after initial O-glycosylation.NThe mucosal surfaces comprise a 1000-fold larger interface between the external and internal milieu than the skin. At the same time, the chemical and microbiological challenges are more demanding. The most important protective component of the mucosal surfaces is the mucus layer, the gel properties of which are due to macromolecules called mucins. A majority of the mucins known today belong to this classical gel-forming type, although a few glycoproteins defined as mucins are membrane-bound with yet unknown physiological functions (1). The present definition of mucin includes all glycoproteins that consist of more than 50% O-linked oligosaccharides and that have a majority of these oligosaccharides localized to mucin domains. These domains have a high number of O-glycosylated Ser and Thr amino acids, often appearing in tandem repeat sequences. Gel-forming mucins are probably altered in several diseases. Thus, alterations in the mucus barrier are probably essential in the pathogenesis of, for example, infections, peptic ulcers, and inflammatory bowel disease. Diseases such as cystic fibrosis and chronic bronchitis, and also trivial infections, are characterized by increased mucus viscosity. Despite its medical interest, the biochemical nature of these altered mucin properties is still poorly understood, largely due to the difficulties associated with the large size of these molecules.The gel-forming mucins are proposed to be disulfide bondstabilized linear polymers of highly glycosylated proteins (2), although other models are discussed (3). A typical example of such a mucin is encoded by the MUC2 gene, one of the few mucin genes fully sequenced (4). The MUC2 mucin occurs in small and large intestine (5, 6) and probably also in the airways upon epithelial stress, such as infection (7). The primary translation product, the mucin apoprotein, has a size of about 600 kDa, including N-glycans. It is composed of five major regions; three of these, one N-terminal, one C-terminal, and one central, are rich in Cys, whereas the two others are rich in Thr, Ser, and Pro. The two latter regions are th...

Rabbit antiserum against a synthetic peptide corresponding to a tandemly repeated amino acid sequence in the human intestinal mucin apoprotein MUC2 was used in immunoprecipitation to study the biosynthesis of MUC2 in the colon-carcinoma cell line LS 174T. Under non-reducing conditions, two bands were precipitated, the smaller with an apparent size of about 700 kDa on SDS/PAGE. When analysed by two-dimensional electrophoresis after reduction, the larger band migrated to the same position as the smaller band and was interpreted as a putative disulphide-bond-stabilized dimer. Pulse-chase experiments showed only the monomer after 5 min and the appearance of the putative dimer after 30 min. The MUC2 apoprotein was also precipitated by antisera against the HF-deglycosylated peptides of the two highly glycosylated domains of the 'insoluble' mucin complex of rat small intestine [Carlstedt, Herrmann, Karlsson, Sheehan, Fransson and Hansson (1993) J. Biol. Chem. 268, [18771-18781]. Endoprotease Lys-C cleavage of the immunopurified apoprotein gave a large fragment of about 250 kDa that was detected by both the antiserum against the MUC2 tandem repeat and one of the glycopeptide antisera. This supports the view that the 'insoluble' mucin of rat small intestine is encoded by the Muc2 gene, as recently indicated by a partial cDNA sequence [Hansson, Baeckström, Carlstedt and Klinga-Levan (1994) Biochem. Biophys. Res. Commun. 198, 181-190] and that parts of the apoprotein are conserved between the species. A lectin from the snail Helix pomatia that detects terminal alpha-GalNAc residues did not bind to the monomer or putative dimer, suggesting that O-glycosylation starts after dimerization. The results indicate that the biosynthetic pathway of the MUC2 mucin may be similar to that of the von Willebrand factor with which MUC2 shares sequence similarities at its C- and N-termini.

The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine-and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin κ-chain signal sequence to direct the protein to the secretory pathway. Pulse-chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bondstabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The Cterminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2.

Background: The teleost Zoarces viviparus (eelpout) lives along the coasts of Northern Europe and has long been an established model organism for marine ecology and environmental monitoring. The scarce information about this species genome has however restrained the use of efficient molecular-level assays, such as gene expression microarrays.