Noomi Asker

A New Method for Large Scale Isolation of Kidney Glomeruli from Mice

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- micro 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.

Endothelium-specific platelet-derived growth factor-B ablation mimics diabetic retinopathy

Loss of pericytes from the capillary wall is a hallmark of diabetic retinopathy, however, the pathogenic significance of this phenomenon is unclear. In previous mouse gene knockout models leading to pericyte deficiency, 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 sufficient to cause retinopathy in mice, implying that pericyte loss may also be a causal pathogenic event in human diabetic retinopathy.

Microarray analysis of blood microvessels from PDGF-B and PDGF-Rβ mutant mice identifies novel markers for brain pericytes

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 pericytedeficient brain microvessels isolated from platelet‐derived growth factor (PDGF‐B) –/– and PDGF beta receptor (PDGFRβ) –/– mouse mutants. The approach was validated by the identification of known pericyte markers among the most down‐regulated genes in PDGF‐B –/– and PDGFRβ –/– microvessels. Of candidates for novel pericyte markers, we selected ATP‐sensitive potassiumchannel 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.—Bondjers, C., He, L., Takemoto, M., Norlin, J., Asker, N., Hellström, M., Lindahl, P., Betsholtz, C. Microarray analysis of blood microvessels from PDGF‐B and PDGF‐Rβ mutant mice identifies novel markers for brain pericytes. FASEB J. 20, E1005–1013 (2006)

Dimerization of the Human MUC2 Mucin in the Endoplasmic Reticulum Is Followed by a N-Glycosylation-dependent Transfer of the Mono- and Dimers to the Golgi Apparatus

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 anO-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 ofN-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 O-glycosylated 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.

O-Glycosylated MUC2 Monomer and Dimer from LS 174T Cells Are Water-soluble, whereas Larger MUC2 Species Formed Early during Biosynthesis Are Insoluble and Contain Nonreducible Intermolecular Bonds

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, N., Axelsson, M. A. B., Olofsson, S.-O., and Hansson, G. C. (1998) J. Biol. Chem. 273, 18857–18863). Using an antiserum immunoprecipitating O-glycosylated MUC2 mucin, monomers and dimers were shown to occur in soluble form in the lysate of LS 174T cells. The amount of O-glycosylated dimer was small, and no larger species were found even after long chase periods. However, most of the labeled MUC2 mucin was found in pelleted debris of the cell lysate. This insoluble MUC2 mucin was recovered by immunoprecipitation after reduction of disulfide bonds. Analysis by agarose gel electrophoresis revealed two bands, of which the smaller migrated as the O-glycosylated monomer and the larger migrated as the O-glycosylated dimer of the cell lysis supernatant. Mucins insoluble in 6 m guanidinium chloride could also be obtained from LS 174T cells. Such mucins have earlier been found in the small intestine (Carlstedt, I., Herrmann, A., Karlsson, H., Sheehan, J., Fransson, L.-Å., and Hansson, G. C. (1993) J. Biol. 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.

Characterization of the Zoarces viviparus liver transcriptome using massively parallel pyrosequencing

BackgroundThe 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.ResultsIn the present study we present the first comprehensive characterization of the Zoarces viviparus liver transcriptome. From 400,000 reads generated by massively parallel pyrosequencing, more than 50,000 pieces of putative transcripts were assembled, annotated and functionally classified. The data was estimated to cover roughly 40% of the total transcriptome and homologues for about half of the genes of Gasterosteus aculeatus (stickleback) were identified. The sequence data was consequently used to design an oligonucleotide microarray for large-scale gene expression analysis.ConclusionOur results show that one run using a Genome Sequencer FLX from 454 Life Science/Roche generates enough genomic information for adequate de novo assembly of a large number of genes in a higher vertebrate. The generated sequence data, including the validated microarray probes, are publicly available to promote genome-wide research in Zoarces viviparus.

The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells

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 kappa-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-bond-stabilized 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 C-terminus 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.

Combination of reverse and chemical genetic screens reveals angiogenesis inhibitors and targets.

We combined reverse and chemical genetics to identify targets and compounds modulating blood vessel development. Through transcript profiling in mice, we identified 150 potentially druggable microvessel-enriched gene products. Orthologs of 50 of these were knocked down in a reverse genetic screen in zebrafish, demonstrating that 16 were necessary for developmental angiogenesis. In parallel, 1280 pharmacologically active compounds were screened in a human cell-based assay, identifying 28 compounds selectively inhibiting endothelial sprouting. Several links were revealed between the results of the reverse and chemical genetic screens, including the serine/threonine (S/T) phosphatases ppp1ca, ppp1cc, and ppp4c and an inhibitor of this gene family; Endothall. Our results suggest that the combination of reverse and chemical genetic screens, in vertebrates, is an efficient strategy for the identification of drug targets and compounds that modulate complex biological systems, such as angiogenesis.

Molecular characterization of the large heavily glycosylated domain glycopeptide from the rat small intestinal Muc2 mucin

The largest high-glycosylated domain, glycopeptide A, of the ‘insoluble’ mucin complex of the rat small intestine has earlier been purified and characterized (Carlstedtet al., 1993,J Biol Chem268: 18771–81). A rabbit antiserum raised against deglycosylated glycopeptide A was used to clone part of a mucin showing homology to the human MUC2 mucin (Hanssonet al., 1994,Biochem Biophys Res Commun198: 181–90). This serum specifically stained goblet cells (paranuclear) in the mouse small intestine. The size of the coding sequence of glycopeptide A was estimated by using reversed transcriptase PCR of mRNA from an inbred rat strain (GOT-W) using primers in the unique central and C-terminal parts of the proposed rat Muc2 sequences. The PCR and Southern blot of the PCR products showed a fragment of about 5.5 kb corresponding to about 1700 amino acids when the known Cys-rich sequences used for the primers were subtracted. This is slightly larger than the size estimated earlier by biochemical studies. The mRNA encoding the rat Muc2 was slightly smaller than the mRNA encoding the human MUC2 in a colorectal cell line. Although the size of glycopeptide A estimated from biochemical results and by PCR is not identical, the results obtained here further support that the ‘insoluble’ mucin of the rat small intestine is encoded by the Muc2 gene. Most of the oligosaccharides in glycopeptide A were either neutral (40%) or sialylated (40%). The remaining ones were sulfated with the sulfate group attached to C-6 ofN-acetylglucosamine linked to C-6 of theN-acetylgalactosaminitol as revealed by tandem mass spectrometry of the perdeuteroacetylated oligosaccharides. Eighteen oligosaccharides were found of which fourteen were characterized and found to be mostly novel. Our findings thus expand the current knowledge of the core peptide of the rat intestinal goblet cell mucin and provide a relatively complete picture of the glycosylation of a defined mucin domain.

Hepatic transcriptome profiling indicates differential mRNA expression of apoptosis and immune related genes in eelpout (Zoarces viviparus) caught at Göteborg harbor, Sweden.

The physiology and reproductive performance of eelpout (Zoarces viviparus) have been monitored along the Swedish coast for more than three decades. In this study, transcriptomic profiling was applied for the first time as an exploratory tool to search for new potential candidate biomarkers and to investigate possible stress responses in fish collected from a chronically polluted area. An oligonucleotide microarray with more than 15,000 sequences was used to assess differentially expressed hepatic mRNA levels in female eelpout collected from the contaminated area at Göteborg harbor compared to fish from a national reference site, Fjällbacka. Genes involved in apoptosis and DNA damage (e.g., SMAC/diablo homolog and DDIT4/DNA-damage-inducible protein transcript 4) had higher mRNA expression levels in eelpout from the harbor compared to the reference site, whereas mRNA expression of genes involved in the innate immune system (e.g., complement components and hepcidin) and protein transport/folding (e.g., signal recognition particle and protein disulfide-isomerase) were expressed at lower levels. Gene Ontology enrichment analysis revealed that genes involved biological processes associated with protein folding, immune responses and complement activation were differentially expressed in the harbor eelpout compared to the reference site. The differential mRNA expression of selected genes involved in apoptosis/DNA damage and in the innate immune system was verified by quantitative PCR, using the same fish in addition to eelpout captured four years later. Thus, our approach has identified new potential biomarkers of pollutant exposure and has generated hypotheses on disturbed physiological processes in eelpout. Despite a higher mRNA expression of genes related to apoptosis (e.g., diablo homolog) in eelpout captured in the harbor there were no significant differences in the number of TUNEL-positive apoptotic cells between sites. The mRNA level of genes involved in apoptosis/DNA damage and the status of the innate immune system in fish species captured in polluted environments should be studied in more detail to lay the groundwork for future biomonitoring studies.