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Biomedical subjects

R Timpl

Publications and source records attributed to R Timpl.

At least 271 records · Page 15Linked to original sources
PubMed1986

Purification and tissue distribution of a small protein (BM-40) extracted from a basement membrane tumor.

A novel acidic glycoprotein, BM-40, with Mr = 40,000, was purified from the basement-membrane-producing mouse EHS tumor and characterized with regard to its unique chemical and antigenic properties. It was obtained from the tumor in a neutral salt-soluble form or as a component requiring extraction with 6M guanidine X HCl. This protein could also be identified in many other tissue extracts and cell and tissue cultures. The most intact form of BM-40 consists of a single polypeptide chain which undergoes limited proteolysis during extraction and purification. BM-40 exists in most tissues in stoichiometric amounts compared to other basement membrane proteins (laminin, nidogen) and is secreted by various teratocarcinoma and epithelial cells. It can be visualized by immunofluorescence in the extracellular matrix of the EHS tumor and Reichert's membrane. Other tissues which contain extractable BM-40 were negative in immunofluorescence.

Animals↗
PubMed1986

Structure of mouse type IV collagen. Amino-acid sequence of the C-terminal 511-residue-long triple-helical segment of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain.

The sequence of 511 residues from the C-terminal portion of the triple helix of mouse alpha 2(IV) chain was determined by using the pepsin fragment P2 of collagen IV and two cDNA clones selected from an Engelbreth-Holm-Swarm (EHS) tumor library. The sequence contains nine interruptions of the triplet repeat Gly-Xaa-Yaa ranging in size from single insertions or deletions up to stretches of eleven amino acid residues. Five of these interruptions match those present in the homologous segment of the alpha 1(IV) chain but are otherwise different in length and/or sequence. A low homology was found for the triplet regions of the alpha 1(IV) and alpha 2(IV) chain which constitute more than 90% of the sequence. The data indicate a remote evolutionary relationship of the triple-helical sequences of the two constituent chains of basement membrane collagen.

Amino Acid Sequence↗
PubMed1986

Purification and structural characterization of intact and fragmented nidogen obtained from a tumor basement membrane.

Extraction of a basement-membrane-producing mouse tumor with 6 M guanidine/HCl in the presence of protease inhibitors allowed the purification of the genuine form of the matrix protein nidogen (Mr = 150,000) and, in addition, two defined fragments (Mr = 130,000 and 100,000). Smaller fragments (Mr = 80,000 and 40,000) were obtained under conditions with less stringent control of endogenous proteolysis. Intact nidogen and the larger fragments were similar in amino acid and carbohydrate (about 5%) composition, the presence of a single polypeptide chain, conformational features as revealed by CD spectroscopy and all shared major epitopes located on the Mr = 80,000 fragment. Additional epitopes were found on intact nidogen and the Mr = 130,000 fragment. Nidogen and the various fragments possess different N-terminal amino acid sequences indicating a stepwise degradation from the N-terminal end of the molecule. Electron microscopical and hydrodynamic studies of the Mr = 80,000 fragment demonstrated a structure consisting of a globular head connected to a thin tail. Intact nidogen appears to contain a somewhat larger globule but the same tail, which is terminated at its opposite end by a second, smaller globular structure. The data suggest a multidomain structure for nidogen containing sites highly susceptible to proteolytic cleavage.

Amino Acids↗
PubMed1986

Structure and function of basement membrane proteoglycans.

Basement membranes contain at least three different proteoglycans. These are a large, low buoyant density heparan sulphate proteoglycan and two smaller, high density proteoglycans with either heparan sulphate or chondroitin sulphate side-chains. The large (Mr 400K-600K and small (Mr 130K) heparan sulphate proteoglycans were purified from the mouse EHS tumour. These proteoglycans are immunologically related by sharing some protein core antigenic determinants (epitopes) but do not cross-react with cell-surface heparan sulphate proteoglycans or with proteoglycans from interstitial connective tissue. This indicates that they belong to a distinct family of proteoglycans. Structural models were developed, based on electron microscopy and analytical ultracentrifugation, demonstrating that the small proteoglycan contains on average four heparan sulphate chains of about 30 nm in length, while the large proteoglycan consists of three long (about 90 nm) heparan sulphate chains connected to one end of a large core protein. Single heparan sulphate chains were isolated from the EHS tumour proteoglycans and from the corresponding proteoglycans from Reichert's membrane of the mouse embryo. The heparan sulphate from Reichert's membrane bound to antithrombin with high affinity and was found to contain the unique 3-O-sulphated glucosamine residue previously identified in the antithrombin-binding region of heparin. The EHS tumour heparan sulphate showed a higher N-/O-sulphate ratio and a lower affinity for antithrombin.

Animals↗
PubMed1986

Immunofluorescent localization of structural collagen types in endochondral fracture repair.

A nonimmobilized rat tibial fracture model of endochondral osseous repair was examined for the unique localizations of specific collagen genetic types. At various stages of the healing process, the demineralized callus was reacted with immunofluorescent antibodies directed against the type specific forms of matrix collagen. Type III collagen rapidly appeared (day 8-10) and remained in the primitive mesenchymal callus until remodeled. It was particularly prominent in the highly vasoformative regions and the pericallus encapsulation but not present in preexisting cortical and neoformed lamellar bone. The type II collagen, a marker of cartilage, was uniquely located only in areas of chondroid differentiation and calcification. Type II collagen was absent from all bone and was not identified beneath the repairing intact periosteum. The differentiating chondrocytes synthesized type II collagen on an underlayer of type III collagen already within the mesenchymal matrix. From these studies of genetically unique collagen markers, it appears that only in areas of motion or anoxia does an intermediate of chondroid tissue appear. The utilization of specific type II and type III collagen immunofluorescent antibodies has facilitated the understanding of the fracture repair process and has acted as an indicator for unique matrix components.

Animals↗
PubMed1986

Light microscopic immunolocalization of laminin, type IV collagen, nidogen, heparan sulphate proteoglycan and fibronectin in the enteric nervous system of rat and guinea pig.

The localization of the extracellular matrix components laminin, fibronectin and type IV collagen in the enteric nervous system and the surrounding smooth muscle was investigated by immunohistochemical methods, using tissue sections of rat and guinea pig large intestine. None of these molecules were detectable inside the enteric ganglia. In contrast, they were easily demonstrable in association with the basement membrane of satellite cells within sensory and sympathetic ganglia. All of these molecules were, however, present in or nearby the basement membrane that surrounds each enteric ganglion. This agrees with previous ultrastructural observations that, in small mammals, neither basement membranes nor large connective tissue spaces are found inside enteric ganglia. The matrix molecules under study were also detected in the basement membrane of the nearby smooth muscle cells that make up the muscle layer of the gut wall. Fibronectin was frequently observed as a broad staining pattern suggesting its localization in the lamina reticularis rather than in the lamina densa. In addition, nidogen and heparan sulphate proteoglycan were demonstrated in the basement membrane of both enteric ganglia and Schwann cells.

Animals↗
PubMed1986

Role of laminin and fibronectin in selecting myogenic versus fibrogenic cells from skeletal muscle cells in vitro.

Growth of embryonic skeletal muscle occurs by fusion of multinucleated myotubes with differentiated, fusion-capable myoblasts. Selective recognition seems to prevent fusion of myotubes with nonmyogenic cells such as muscle fibroblasts, endothelial cells, or nerve cells, but the nature of the signal is as yet unknown. Here we provide evidence that one of the selection mechanisms may be the enhanced affinity for laminin of myogenic cells as compared to fibrogenic cells. Growing myotubes in myoblast cultures accumulate laminin and type IV collagen on their surface in patches and strands as the first step in assembling a continuous basal lamina on mature myofibers (U. Kühl, R. Timpl, and K. von der Mark (1982), Dev. Biol. 93, 344-359). Fibronectin, on the other hand, assembles into an intercellular fibrous meshwork not associated with the free myotube surface. Over a brief time period (10-20 min) myoblasts from embryonic mouse thigh muscle adhere faster to laminin than do fibroblasts from the same tissue; these adhere faster to fibronectin. When a mixture of the cells is plated for 20 min on laminin/type IV collagen substrates, only myogenic cells adhere, giving rise to cultures with more than 90% fusion after 2 weeks; on fibronectin/type I collagen in the same time primarily fibroblastic cells adhere, giving rise to cultures with less than 10% nuclei in myotubes. The differential affinities of myoblasts for basement membrane constituents and of fibroblasts for interstitial connective tissue components may play a role in sorting out myoblasts from fibroblasts in skeletal muscle development.

Animals↗
PubMed1986

Defective attachment of dermatosparactic fibroblasts to collagens I and IV.

Attachment of fibroblasts from dermatosparactic sheep and cattle to collagenous substrates (types I and IV) is defective (30-50%) when compared with fibroblasts from normal or heterozygous animals. The difference was independent of the amount of substrate, incubation time and protein synthesis. No differences were observed in the binding to fibronectin or laminin. Reduced attachment to collagen can be partially restored by adding fibronectin. The polygonal morphology of dermatosparactic cells was, however, not altered by attachment and growth on dishes coated with different collagens or fibronectin. Reduced interaction with collagens could be due to changes in specific receptors and may represent a further pathological change in dermatosparactic animals.

Animals↗
PubMed1986

Laminin increases both levels and activity of tyrosine hydroxylase in calf adrenal chromaffin cells.

We have investigated the effects of substrate-bound laminin on levels of enzymes of the catecholamine biosynthetic pathway in primary cultures of calf adrenal chromaffin cells. Laminin increases the levels of the enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyl-transferase. This effect is selective, in that levels of other enzymes (lactate dehydrogenase, aromatic amino acid decarboxylase, and acetylcholinesterase) are not increased. The effect of laminin can be blocked by antibodies directed against a fragment of the heparin-binding domain of the molecule, whereas antibodies directed against other fragments do not block the increase in tyrosine hydroxylase. Thus the laminin domain involved in enzyme regulation in chromaffin cells is apparently the same as that previously implicated in laminin's interactions with neurons to potentiate survival and stimulate neurite outgrowth (Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468). The increase in chromaffin cell tyrosine hydroxylase levels is preceded by an activation of the enzyme in which the Vmax (but not the Km) is altered. The effects of laminin appear to be developmentally regulated, since neither activation nor increased levels of tyrosine hydroxylase occur in adult adrenal chromaffin cells exposed to laminin.

Animals↗
PubMed1986

Type VI collagen in extracellular, 100-nm periodic filaments and fibrils: identification by immunoelectron microscopy.

Filaments and fibrils that exhibit a 100-nm axial periodicity and occur in the medium and in the deposited extracellular matrix of chicken embryo and human fibroblast cultures have been tentatively identified with type VI collagen on the basis of their similar structural characteristics (Bruns, R. R., 1984, J. Ultrastruct. Res., 89:136-145). Using indirect immunoelectron microscopy and specific monoclonal and polyclonal antibodies, we now report their positive identification with collagen VI and their distribution in fibroblast cultures and in tendon. Primary human foreskin fibroblast cultures, labeled with anti-type VI antibody and studied by fluorescence microscopy, showed a progressive increase in labeling and changes in distribution with time up to 8 d in culture. With immunoelectron microscopy and monoclonal antibodies to human type VI collagen followed by goat anti-mouse IgG coupled to colloidal gold, they showed in thin sections specific 100-nm periodic labeling on extracellular filaments and fibrils: one monoclonal antibody (3C4) attached to the band region and another (4B10) to the interband region of the filaments and fibrils. Rabbit antiserum to type VI collagen also localized on the band region, but the staining was less well defined. Control experiments with antibodies to fibronectin and to procollagen types I and III labeled other filaments and fibrils, but not those with a 100-nm period. Heavy metal-stained fibrils with the same periodic and structural characteristics also have been found in both adult rat tail tendon and embryonic chicken tendon subjected to prolonged incubation in culture medium or treatment with adenosine 5'-triphosphate at pH 4.6. We conclude that the 100-nm periodic filaments and fibrils represent the native aggregate form of type VI collagen. It is likely that banded fibrils of the same periodicity and appearance, reported by many observers over the years in a wide range of normal and pathological tissues, are at least in part, type VI collagen.

Adenosine Triphosphate↗
PubMed1986

Attachment of cells to basement membrane collagen type IV.

Of ten different cell lines examined, three showed distinct attachment and spreading on collagen IV substrates, and neither attachment nor spreading was enhanced by adding soluble laminin or fibronectin. This reaction was not inhibited by cycloheximide or antibodies to laminin, indicating a direct attachment to collagen IV without the need of mediator proteins. Cell-binding sites were localized to the major triple-helical domain of collagen IV and required an intact triple helical conformation for activity. Fibronectin showed preferential binding to denatured collagen IV necessary to mediate cell binding to the substrate. Fibronectin binding sites of collagen IV were mapped to unfolded structures of the major triple-helical domain and show a similar specificity to fibronectin-binding sites of collagen I. The data extend previous observations on biologically potential binding sites located in the triple helix of basement membrane collagen IV.

Animals↗
PubMed1986

Type III collagen aminopropeptide levels in serum of patients with progressive systemic scleroderma.

Sera from 101 patients with progressive systemic scleroderma were analyzed for circulating aminopropeptides of type III collagen using a radioimmunoassay which measures the intact and degraded forms (Fab assay). About 41% of the patients were found to have values above the normal range. A good correlation was observed between elevated levels of aminopropeptides and the degree of involvement of the skin and internal organs in the patients. Most patients (89%) with an active progression of the disease but not those in a stationary phase showed increased serum levels of aminopropeptides. Treatment with corticosteroids apparently normalized the levels of aminopropeptides. Only minor changes were observed with an antibody-based radioimmunoassay which measures primarily the intact form of the aminopropeptide.

Adult↗
PubMed1986

Immunochemistry of elastotic material in sun-damaged skin.

The nature of elastotic material in sun-damaged human skin was investigated by indirect immunofluorescence. Antibodies were used against the following components of the dermis: type I and type VI collagens, aminopropeptide of type I and type III procollagens, fibronectin, elastin, microfibrillar proteins, and basement membrane represented by the 7S domain of type IV collagen, laminin, and nidogen. The elastotic material exhibited marked fluorescence for elastin and microfibrillar proteins which codistributed with fibronectin. The presence of type I and VI collagens and procollagen type III were demonstrated to a lesser extent within the elastotic material. These results suggest that solar elastosis is primarily derived from elastic fibers and not from preexisting or newly synthesized collagens.

Antibodies↗
PubMed1986

Basement membrane proteins produced by Schwann cells and in neurofibromatosis.

Mouse Schwann cells and rat RN22 schwannoma cells cultured in the absence of neurons and fibroblasts produce typical basement membrane proteins. Heparan sulfate proteoglycan (low density form), nidogen, and protein BM-40 were identified by radioimmunoassays, immunoblotting, and by immunoprecipitation after metabolic labeling. The cells also produce a laminin-like protein that differs from authentic laminin by a reduced A chain content and lack of antigenic determinants located in the long arm of laminin. Laminin possessing A and B chains is, however, produced by PYS-2 teratocarcinoma cells grown under the same conditions. Laminin from Schwann cell culture medium promotes neurite outgrowth, and this activity could be immunoprecipitated but not blocked by various antibodies against authentic laminin. In addition, Schwann cell laminin is found complexed noncovalently with nidogen. Sulfate incorporation revealed the synthesis of proteoglycans and entactin. A similar set of proteins and in addition collagen IV could be demonstrated in neurofibroma tissue by immunohistology, and were localized to the laminae densae of the multilayered basement membranes around Schwann cells and capillaries. Laminin purified from 0.5 M NaCl neurofibroma tissue extracts possessed both A and B chains. Nidogen was identified in a partially degraded form.

Animals↗
PubMed1986

Binding of collagen to Staphylococcus aureus Cowan 1.

Collagen binds to a receptor protein present on the surfaces of Staphylococcus aureus cells. Binding of 125I-labeled type II collagen to its bacterial receptor is reversible, and Scatchard plot analysis indicates the presence of one class of receptor that occurs on an average of 3 X 10(4) copies per cell and binds type II collagen with a Kd of 10(-7) M. Studies on the specificity of collagen cell binding indicate that the receptor does not recognize noncollagenous proteins but binds all of the different collagen types tested (types I to VI). Furthermore, isolated collagen alpha chains and peptides generated by cyanogen bromide cleavage of type I collagen alpha chains are recognized by the receptor as indicated by the ability of these polypeptides to inhibit binding of 125I-labeled type II collagen to staphylococcal cells. Synthetic collagen analogs were tested as inhibitors of type II collagen binding to bacterial cells. The peptides (Pro-Gly-Pro)n, (Pro-Pro-Gly)10, and (Pro-OH-Pro-Gly)10 were recognized by the receptor, whereas the peptides (Pro-Ala-Gly)n and polyproline showed no inhibitory activity.

Collagen↗
PubMed1986

Antibodies to basement membrane protein nidogen in Chagas' disease and American cutaneous leishmaniasis.

About 50 to 70% of sera from patients with American cutaneous leishmaniasis and chronic Chagas' disease possessed antibodies which reacted in enzyme and radioimmunoassays with nidogen obtained from a tumor basement membrane. The antibodies were of the immunoglobulin M and G classes in acute American cutaneous leishmaniasis but mainly of the immunoglobulin G class in chronic Chagas' disease. Similar antibodies could not be detected in patients suffering from a variety of other infectious or inflammatory diseases when compared with healthy control groups. Inhibition and immunoadsorption studies indicated a close relationship of epitopes recognized by patients' antibodies on nidogen and on another basement membrane protein, laminin. Since rabbit antisera to both proteins do not cross-react, a special nature of the epitopes involved in the reaction with patient sera is suggested. Similar epitopes may exist on various forms of Leishmania or Trypanosoma protozoa.

Antibodies↗