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M Paulsson

Publications and source records attributed to M Paulsson.

At least 37 records · Page 2Linked to original sources
PubMed2000

Molecular structure and tissue distribution of matrilin-3, a filament-forming extracellular matrix protein expressed during skeletal development.

Matrilin-3 is a recently identified member of the superfamily of proteins containing von Willebrand factor A-like domains and is able to form hetero-oligomers with matrilin-1 (cartilage matrix protein) via a C-terminal coiled-coil domain. Full-length matrilin-3 and a fragment lacking the assembly domain were expressed in 293-EBNA cells, purified, and subjected to biochemical characterization. Recombinantly expressed full-length matrilin-3 occurs as monomers, dimers, trimers, and tetramers, as detected by electron microscopy and SDS-polyacrylamide gel electrophoresis, whereas matrilin-3, purified from fetal calf cartilage, forms homotetramers as well as hetero-oligomers of variable stoichiometry with matrilin-1. In the matrix formed by cultured chondrosarcoma cells, matrilin-3 is found in a filamentous, collagen-dependent network connecting cells and in a collagen-independent pericellular network. Affinity-purified antibodies detect matrilin-3 expression in a variety of mouse cartilaginous tissues, such as sternum, articular, and epiphyseal cartilage, and in the cartilage anlage of developing bones. It is found both inside the lacunae and in the interterritorial matrix of the resting, proliferating, hypertrophic, and calcified cartilage zones, whereas the expression is lower in the superficial articular cartilage. In trachea and in costal cartilage of adult mice, an expression was seen in the perichondrium. Furthermore, matrilin-3 is found in bone, and its expression is, therefore, not restricted to chondroblasts and chondrocytes.

Animals↗
PubMed2000

Structure and mapping of the mouse matrilin-3 gene (Matn3), a member of a gene family containing a U12-type AT-AC intron.

The gene for murine matrilin-3, an extracellular matrix protein present in cartilage, was isolated and further characterized. The gene spans 23.4 kb and comprises 8 exons; with one exception, this reflects the modular structure of the protein. The major and a minor transcription start site were determined by RNase protection assays to positions approximately 72 nt and 87 nt upstream of the ATG codon, respectively. The promoter contains a TATA-like box 32 bp upstream of the main transcription start as well as several potential binding sites for eukaryotic transcription factors. As in all known matrilin genes, the last intron, separating the exons coding for the coiled-coil domain, does not follow the GT-AG rule and belongs to the subgroup of introns having AT-AC at the ends that are spliced by the U12-type spliceosome. The mouse matrilin-3 gene does not contain hidden exon sequences coding for the second vWFA-like domain present in all other matrilins. The intron that could possibly contain such sequences instead shows 75% repetitive sequences, indicating an evolutionary process that has led to the loss of sequences coding for vWFA2. Single-strand conformation polymorphism analysis was used to map the Matn3 gene to the proximal end of Chr 12, linked to the genes Synd1, Apob, Dntb, and Kif3c.

Amino Acid Sequence↗
PubMed2000

Evaluation of mucoadhesion for two polyelectrolyte gels in simulated physiological conditions using a rheological method.

A rheological method to measure mucoadhesion was evaluated for two ion-sensitive polymers, Carbopol 934 and Gelrite((R)) (deacetylated gellan gum), in a simulated physiological environment using two commercially available mucins. The method simulates the interpenetration layer in the mucoadhesion process. The elastic modulus for a polymer/mucin mixture is compared with the elastic modulus for the polymer alone, and an increase in the elastic modulus for the mixture compared to the polymer is interpreted as a positive interaction caused by mucoadhesion. In this study the influence of polymer concentration, type of mucin and experimental rheological factors, such as gap width, were examined. The choice of polymer concentration was crucial, especially with the porcine gastric mucin. We found that one is more likely to obtain positive interactions with weak gels. It was also shown that the choice of mucin has a large influence on the results obtained. Carbopol 934 interacted more strongly with the bovine submaxillary gland mucin than with the porcine gastric mucin, whereas the gel structure of Gelrite((R)) was destroyed when mixed with the bovine mucin. Furthermore, it was concluded that with hydrogels consisting of gel particles (such as Carbopol 934), rheological measurements can give highly varying results, depending on, for example, the concentration and ion-sensitivity of the polymer, the quantity of ions present, as well as the gap width of the measuring system.

Acrylic Resins↗
PubMed2000

Expression of matrilin-1, -2 and -3 in developing mouse limbs and heart.

The expression of matrilin-1, -2 and -3 was studied in the heart and limb during mouse development. Matrilin-1 is transiently expressed in the heart between days 9.5 and 14.5 p.c. Matrilin-2 expression was detected in the heart from day 10.5 p.c. onwards. In the developing limb bud, both matrilin-1 and -3 were observed first at day 12.5 p.c. Throughout development matrilin-3 expression was strictly limited to cartilage, while matrilin-1 was also found in some other forms of connective tissue. Matrilin-2, albeit present around hypertrophic chondrocytes in the growth plate, was mainly expressed in non-skeletal structures. The complementary, but in part overlapping, expression of matrilins indicates the possibility for both redundant and unique functions among the members of this novel family of extracellular matrix proteins.

Animals↗
PubMed2000

The absence of nidogen 1 does not affect murine basement membrane formation.

Nidogen 1 is a highly conserved protein in mammals, Drosophila melanogaster, Caenorhabditis elegans, and ascidians and is found in all basement membranes. It has been proposed that nidogen 1 connects the laminin and collagen IV networks, so stabilizing the basement membrane, and integrates other proteins, including perlecan, into the basement membrane. To define the role of nidogen 1 in basement membranes in vivo, we produced a null mutation of the NID-1 gene in embryonic stem cells and used these to derive mouse lines. Homozygous animals produce neither nidogen 1 mRNA nor protein. Surprisingly, they show no overt abnormalities and are fertile, their basement membrane structures appearing normal. Nidogen 2 staining is increased in certain basement membranes, where it is normally only found in scant amounts. This occurs by either redistribution from other extracellular matrices or unmasking of nidogen 2 epitopes, as its production does not appear to be upregulated. The results show that nidogen 1 is not required for basement membrane formation or maintenance.

Animals↗
PubMed2000

All three PACSIN isoforms bind to endocytic proteins and inhibit endocytosis.

The PACSINs are a family of cytoplasmic phosphoproteins that play a role in vesicle formation and transport. We report the cloning and cDNA sequencing of PACSIN 3 and the analysis of all three PACSIN isoforms with regard to tissue distribution, ligand binding properties and influence on endocytosis. PACSIN 3 differs from the other family members in having a short proline-rich region and lacking asparagine-proline-phenylalanine motifs. In contrast to the neurospecific PACSIN 1 and the ubiquitously expressed PACSIN 2, PACSIN 3 is mainly detected in lung and muscle tissues. All isoforms potentially oligomerize and bind to dynamin, synaptojanin 1 and N-WASP via their Src homology 3 domains. The PACSIN proteins colocalize with dynamin, but not with clathrin, implying a specific role with a distinct subpopulation of dynamin at defined cellular sites. Transferrin endocytosis is blocked in a dose-dependent manner in cells overexpressing the PACSIN variants, but the inhibitory effect can be abolished by mutating specific amino acid residues in the Src homology 3 domains. These characteristics of the PACSIN protein family suggest a general function in recruitment of the interacting proteins to sites of endocytosis.

Adaptor Proteins, Signal Transducing↗
PubMed1999

PACSIN 2, a novel member of the PACSIN family of cytoplasmic adapter proteins.

The PACSIN-related proteins are cytoplasmic adapter proteins with a common arrangement of domains and conserved regions. Here we report the cloning, sequencing, and expression of PACSIN 2, a novel member of the PACSIN protein family and accordingly rename the original PACSIN to PACSIN 1. The sequences of the murine and human cDNAs reveal an open reading frame encoding a putative protein of 486 residues. Despite its high sequence similarity to PACSIN 1, PACSIN 2 is encoded by distinct transcripts in human and mouse, in particular displaying a ubiquitous expression pattern. Immunofluorescence microscopy of PACSIN 2-transfected NIH3T3 fibroblasts reveal a broad, vesicle-like cytoplasmic staining. In contrast to FAP52, another PACSIN-related protein derived from chicken brain, PACSIN 2 could not be detected at focal contacts. Taken together, these findings suggest that PACSIN 2 is a novel PACSIN isoform with similar domain and motif arrangement, but an unrestricted expression pattern, which may participate in the organization of the actin cytoskeleton and the regulation of vesicular traffic.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Matrilin-2, a large, oligomeric matrix protein, is expressed by a great variety of cells and forms fibrillar networks.

Matrilin-2 is a member of the protein superfamily with von Willebrand factor type A-like modules. Mouse matrilin-2 cDNA fragments were expressed in 293-EBNA cells, and the protein was purified, characterized, and used to immunize rabbits. The affinity-purified antiserum detects matrilin-2 in dense and loose connective tissue structures, subepithelial connective tissue of the skin and digestive tract, specialized cartilages, and blood vessel walls. In situ hybridization of 35S-labeled riboprobes localizes the matrilin-2 mRNA to fibroblasts of dermis, tendon, ligaments, perichondrium, and periosteum; connective tissue elements in the heart; smooth muscle cells; and epithelia and loose connective tissue cells of the alimentary canal and respiratory tract. RNA blot hybridization and immunoblotting revealed both matrilin-2 mRNA and protein in cultures of a variety of cell types, confirming the tissue distribution. Alternative splicing affects a module unique for matrilin-2 in all of the above RNA sources. SDS-polyacrylamide gel electrophoresis and electron microscopy reveals matrilin-2 from tissue extracts and cell line cultures as a mixture of mono-, di-, tri-, and tetramers. Matrilin-2 is substituted with N-linked oligosaccharides but not with glycosaminoglycans. Because of other, yet unidentified, cell-type dependent posttranslational modifications, the monomer is heterogeneous in size. Immunofluorescence showed that matrilin-2 functions by forming an extracellular, filamentous network.

Animals↗
PubMed1999

The intrinsic factor-vitamin B12 receptor, cubilin, is assembled into trimers via a coiled-coil alpha-helix.

A large protein was purified from bovine kidney, using selective extraction with EDTA to solubilize proteins anchored by divalent cation-dependent interactions. An antiserum raised against the purified protein labeled the apical cell surface of the epithelial cells in proximal tubules and the luminal surface of small intestine. Ten peptide sequences, derived from the protein, all matched the recently published sequences for rat (Moestrup, S. K., Kozyraki, R., Kristiansen, M., Kaysen, J. H., Holm Rasmussen, H., Brault, D., Pontillon, F., Goda, F. O., Christensen, E. I., Hammond, T. G., and Verroust, P. J. (1998) J. Biol. Chem. 273, 5235-5242) and human cubilin, a receptor for intrinsic factor-vitamin B12 complexes, identifying the protein as bovine cubilin. In electron microscopy, a three-armed structure was seen, indicating an oligomerization of three identical subunits. This model was supported by the Mr values of about 1,500,000 for the intact protein and 440,000 for its subunits obtained by analytical ultracentrifugation. In a search for a potential assembly domain, we identified a region of heptad repeats in the N-terminal part of the cubilin sequence. Computer-assisted analysis supported the presence of a coiled-coil alpha-helix between amino acids 103 and 132 of the human cubilin sequence and predicted the formation of a triple coiled-coil. We therefore conclude that cubilin forms a noncovalent trimer of identical subunits connected by an N-terminal coiled-coil alpha-helix.

Amino Acid Sequence↗
PubMed1999

Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains.

The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits. The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.

Amino Acid Sequence↗
PubMed1999

Absence of basement membranes after targeting the LAMC1 gene results in embryonic lethality due to failure of endoderm differentiation.

The LAMC1 gene coding for the laminin gamma1 subunit was targeted by homologous recombination in mouse embryonic stem cells. Mice heterozygous for the mutation had a normal phenotype and were fertile, whereas homozygous mutant embryos did not survive beyond day 5.5 post coitum. These embryos lacked basement membranes and although the blastocysts had expanded, primitive endoderm cells remained in the inner cell mass, and the parietal yolk sac did not develop. Cultured embryonic stem cells appeared normal after targeting both LAMC1 genes, but the embryoid bodies derived from them also lacked basement membranes, having disorganized extracellular deposits of the basement membrane proteins collagen IV and perlecan, and the cells failed to differentiate into stable myotubes. Secretion of the linking protein nidogen and a truncated laminin alpha1 subunit did occur, but these were not deposited in the extracellular matrix. These results show that the laminin gamma1 subunit is necessary for laminin assembly and that laminin is in turn essential for the organization of other basement membrane components in vivo and in vitro. Surprisingly, basement membranes are not necessary for the formation of the first epithelium to develop during embryogenesis, but first become required for extra embryonic endoderm differentiation.

Animals↗
PubMed1999

Rheological studies of the gelation of deacetylated gellan gum (Gelrite) in physiological conditions.

Gels have been successfully used to increase the mucosal contact time and hence the bioavailability of nasal and ophthalmic formulations. The use of in situ gelling polymers requires a rapid sol-gel transition that produces a strong gel for an optimal contact time. In this study, the rheological behaviour of deacetylated gellan gum (Gelrite) was analysed in order to better understand the reasons for the good performance in humans. Thermal scans were used to study gel formation and other changes in the structure of the samples when the macromolecular and ionic contents were altered. The effect the different ions in tear fluid (Na+, K+, Ca2+) had on the gel strength and the consequences of dilution due to the ocular protective mechanisms were examined. Na+ was found to be the most important gel-promoting ion in vivo. It was also found that gels are formed in tear fluid even when the concentration of Gelrite) is only 0.1%. Samples with concentrations of Gelrite of 0.5-1% do not require more ions than 10-25% of those in tear fluid to form gels. These two findings can partly explain the good performance of Gelrite in vivo. Gels with a high elastic modulus can thus be formed even though dilution of instilled drops takes place.

Acetylation↗
PubMed1999

The matrilins: a novel family of oligomeric extracellular matrix proteins.

The matrilin family at present has four members that all share a structure made up of von Willebrand factor A domains, epidermal growth factor-like domains and a coiled coil alpha-helical module. The first member of the family, matrilin-1 (previously called cartilage matrix protein or CMP), is expressed mainly in cartilage. Matrilin-3 has a similar tissue distribution, while matrilin-2 and -4 occur in a wide variety of extracellular matrices. Matrilin-1 is associated with cartilage proteoglycans as well as being a component of both collagen-dependent and collagen-independent fibrils and on the basis of the related structures other matrilins may play similar roles. The matrilin genes are strictly and differently regulated and their expression may serve as markers for cellular differentiation.

Animals↗
PubMed1999

Cell adhesion to a population of laminin isoforms isolated from normal renal tissue.

To assess whether cells react differently towards a population of several laminin isoforms, as found in vivo, vs. a single isoform, we have compared the biological activity of kidney laminins to that of pure laminin 1. The kidney laminin preparation contained laminin 1 and further isoforms. Both substrates induced adhesion of a large spectrum of cell types, with kidney laminins being the most active. Unfolding of the coil-coiled conformation of the kidney isoforms negatively affected cell adhesion-promoting activity, which indicated that conformation-dependent cell binding is a characteristic feature of many or all laminins. Cellular interactions with kidney laminins were mediated by alpha3beta1 and alpha6beta1 integrins, with the contribution of alpha3beta1 being apparently lower than that of alpha6beta1 integrins. Immunofluorescence staining of vinculin and integrin subunits decorated focal adhesions on kidney laminins which differed in morphology from those formed on laminin 1 alone, in spite of the presence of the latter in the kidney preparation. These observations collectively indicate that tissue specific but often overlapping expression of laminin isoforms might modulate cell behavior by the activation of distinct sets of integrins and by the induction of distinct molecular assemblies within the cell adhesion signaling complexes.

Amino Acid Sequence↗
PubMed1999

Colloidal and thermal characteristics of concentrated dispersions of polymethacrylate-based latices for aqueous enteric coating.

We have used rheological and thermal methods to study the colloidal characteristics of a widely used technical latex. The dispersions of poly(methacrylic acid-ethyl acrylate) (Eudragit L100-55) were found to be stabilized by a combination of electrostatic and steric mechanisms termed as electrosteric stabilization. The electrosteric stabilization is considered to arise in part from dissolved polymer chains with charged carboxylic groups extending out into the continuous phase. The presence of dissolved polymer chains in the dispersion implies that coalescence and interpenetration will be facilitated during film formation, enabling a smooth continuous film to be formed. The extent of the stabilization layer and an effective hard-sphere volume was estimated to discuss the steady shear and viscoelastic properties in this context. The glass transition temperature (Tg) of the particles making up the dispersion has also been determined as a function of sorbed moisture and modeled by the Gordon-Taylor equation modified for specific interaction between water, surfactant, and polymer. This parameter at high moisture content can be used as a first approximation to the minimum film-forming temperature (MFT). Change in Tg (and thus MFT) with moisture content implies that the coating process must be controlled so as to produce a rate of drying slow enough to allow coalescence to occur.

Adsorption↗
PubMed1999

Molecular cloning of testican-2: defining a novel calcium-binding proteoglycan family expressed in brain.

We have screened a human cDNA library using an expressed sequence tag related to the BM-40/secreted protein, acidic and rich in cysteine (SPARC)/osteonectin family of proteins and isolated a novel cDNA. It encodes a protein precursor of 424 amino acids that consists of a signal peptide, a follistatin-like domain, a Ca2+-binding domain, a thyroglobulin-like domain, and a C-terminal region with two putative glycosaminoglycan attachment sites. The protein is homologous to testican-1 and was termed testican-2. Testican-1 is a proteoglycan originally isolated from human seminal plasma that is also expressed in brain. Northern blot hybridization of testican-2 showed a 6.1-kb mRNA expressed mainly in CNS but also found in lung and testis. A widespread expression in multiple neuronal cell types in olfactory bulb, cerebral cortex, thalamus, hippocampus, cerebellum, and medulla was detected by in situ hybridization. A recombinant fragment consisting of the Ca2+-binding EF-hand domain and the thyroglobulin-like domain of testican-2 showed a reversible Ca2+-dependent conformational change in circular dichroism studies. Testican-1 and -2 form a novel Ca2+-binding proteoglycan family built of modular domains with the potential to participate in diverse steps of neurogenesis.

Amino Acid Sequence↗