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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

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↗
PubMed1986

Radioimmunoassay of laminin in serum and its application to cancer patients.

In this sensitive radioimmunoassay for laminin fragment P1 (purified from pepsin extracts of human placenta), monovalent antibody fragments obtained from rabbit antisera against the laminin component are used. The inhibition assay showed a low intra- and interassay variability, and the inhibition curves for various serum samples had parallel slopes. Molecular-sieve chromatography demonstrated heterogeneity of the laminin antigen in serum. For quantification we defined arbitrary units based on the amount of antigenic material present in normal human serum. Sera from 361 tumor patients showed increased values in about half of the cases. Correlation with increased concentration of carcinoembryonic antigen was poor (r = 0.259). The assay may be useful for diseases involving basement-membrane metabolism.

Basement Membrane↗
PubMed1986

Globular domain of basement membrane collagen induces autoimmune pulmonary lesions in mice resembling human Goodpasture disease.

A distinct circulating antibody response could be evoked in C57BL mice after immunization with the globular domain NC1 of basement membrane collagen IV obtained from the mouse Engelbreth-Holm-Swarm tumor when injected together with complete Freund's adjuvant. The antibodies reacted with various subunits of NC1, did not cross-react with other basement membrane proteins, and exhibited a tissue reactivity restricted to certain basement membranes. Tissue-bound antibodies could be detected by direct immunofluorescence and were distributed together with C3 in a linear pattern along glomerular and alveolar basement membranes. Pathological changes were mainly observed in lung and kidney and consisted of inflammatory infiltrates and massive hemorrhages with strong granulomatous fibrotic development in the lung. Kidney alterations were comparably weaker and of focal nature. A nephrotoxic serum model showed rapid binding of rabbit antibodies against mouse NC1 to lung, liver, and kidney basement membranes which was followed several weeks later by an autologous phase with anti-rabbit IgG antibodies bound to basement membranes as immune complexes. There was no fibrotic response but hemorrhagic and inflammatory lung and kidney changes similar to those after active immunization were observed after passive transfer. The experimental NC1 autoimmune model has several features such as anti-NC1 response, tissue restriction, lung hemorrhages, and glomerulonephritis in common with patients suffering from Goodpasture disease. The development of lung fibrosis appears to be unique for the animal model.

Animals↗
PubMed1985

Nature of sulphated macromolecules in mouse Reichert's membrane. Evidence for tyrosine O-sulphate in basement-membrane proteins.

Seven different sulphated macromolecules were detected in 6 M-guanidinium chloride extracts of metabolically [35S]sulphate-labelled mouse Reichert's membrane and were partially separated. Polypeptide bands of apparent Mr 50 000, 150 000 (tentatively identified as entactin) and 170 000 contained essentially tyrosine O-sulphate as the labelled component. Most of the radioactive sulphate was incorporated into three different proteoglycans, which could be separated by chromatography and density-gradient centrifugation before and after enzymic degradation. Enzymic analysis of glycosaminoglycans and of protein cores by immunoassays identified these components as low-density and high-density forms of heparan sulphate proteoglycan and a high-density form of chondroitin sulphate or dermatan sulphate proteoglycan.

Animals↗
PubMed1985

Immunochemical and autoantigenic properties of the globular domain of basement membrane collagen (type IV).

Polyclonal rabbit antibodies raised against the globular domain NC1 of collagen IV from human placenta and a mouse tumor react with conformational antigenic determinants present on the NC1 hexamers and also with the three major subunits obtained after dissociation. The antibodies recognized unique structures within basement membranes and showed a broad tissue reactivity but only limited species cross-reactivity. Using these antibodies, it was possible to detect small amounts of collagen IV antigens from cell cultures and in serum. Monoclonal rat antibodies against mouse NC1 revealed a similar reaction potential. Autoantibodies could be produced in mice against mouse NC1 which react with kidney and lung basement membranes in a pathological manner, mimicking Goodpasture syndrome.

Animals↗
PubMed1985

Collagen synthesis regulation by the aminopropeptide of procollagen I in normal and scleroderma fibroblasts.

The effect of the aminopropeptide of type I procollagen, Col 1(I), on collagen synthesis by normal and scleroderma fibroblasts was investigated. Collagen synthesis was inhibited by about 42% when normal fibroblasts were labeled with 3H-proline in the presence of 4 microM Col 1(I). In contrast, collagen synthesis by scleroderma fibroblasts was inhibited to a lesser degree, about 19%. Furthermore, scleroderma fibroblasts with elevated rates of collagen synthesis (2.5-5 times normal) were inhibited by only about 10%. The data suggest that the increased accumulation of collagen in the skin of scleroderma patients may be related to a defect in the regulation of collagen synthesis by the aminopropeptide.

Adult↗
PubMed1985

Evidence for coiled-coil alpha-helical regions in the long arm of laminin.

Three new laminin fragments, E8, E9 and 25K with mol. wt. 50 000-280 000, were prepared from a limited elastase digest of laminin and from tissue extracts. They were similar with respect to their rod-like structure, a high alpha-helix content, the assembly from two chain segments and immunological cross-reactivity. Two of the fragments (E8 and E9) possess in addition globular domains which lack alpha-helices. Chemical, immunological and physical data together with sequence analysis strongly indicate that the alpha-helical segments are assembled in coiled-coil structures which are located in the rod of the long arm of laminin. These data give new insights into the overall structure of the protein.

Animals↗
PubMed1985

Immunological characterization of basement membrane types of heparan sulfate proteoglycan.

Antibodies were raised against a small high-density and a large low-density form of heparan sulfate proteoglycan from a basement membrane-producing mouse tumor and were characterized by radioimmunoassays, immunoprecipitation and immunohistological methods. Antigenicity was due to the protein cores and included epitopes unique to the low density form as well as some shared by both proteoglycans. The antibodies did not cross-react with other basement membrane proteins or with chondroitin sulfate proteoglycans from interstitial connective tissues. The heparan sulfate proteoglycans occurred ubiquitously in embryonic and adult basement membranes and could be initially detected at the 2-4 cell stage of mouse embryonic development. Low levels were also found in serum. Biosynthetic studies demonstrated identical or similar proteoglycans in cultures of normal and carcinoembryonic cells and in organ cultures of fetal tissues. They could be distinguished from liver cell membrane heparan sulfate proteoglycan, indicating that the basement membrane types of proteoglycans represent a unique class of extracellular matrix proteins.

Animals↗
PubMed1985

Identification and interaction repertoire of large forms of the basement membrane protein nidogen.

Nidogen was purified in its genuine form with a mol. wt. of 150 000 (Nd-150) and as fragments with mol. wts. of 100 000 (Nd-100) and 80 000 (Nd-80) from a mouse tumor basement membrane by preventing activity of endogenous proteases with 6 M guanidine and protease inhibitors. The larger forms of nidogen were also identified in stable complexes with laminin in neutral salt extracts of the tumor and in cell culture medium. Purified Nd-150 and Nd-100, but not Nd-80, were shown to interact with laminin in various binding assays, albeit with lower potential than estimated for the genuine complexes formed in situ. Binding of Nd-150 and Nd-100 to fibronectin and to the globular domain of collagen IV was also observed, but not to heparan sulfate proteoglycan.

Animals↗
PubMed1985

Expression of nidogen and laminin in basement membranes during mouse embryogenesis and in teratocarcinoma cells.

Nidogen and laminin were localized at preimplantation stages of mouse development by immunofluorescence. Laminin was already present on the cell surface at the 2-cell stage, while nidogen was first detectable on compacted 8- to 16-cell stage morulae. Nidogen and laminin colocalized at the blastocyst stage and in postimplantation basement membranes. Immunoblot analyses of tissue extracts and cell culture media indicated the 150-kDa form of nidogen as the largest and predominant form in all tissues examined. Radiolabeled nidogen and laminin synthesized by Reichert's membrane were coprecipitated by antibodies against each antigen, indicating complex formation in situ. Equimolar amounts of laminin and nidogen were determined in 6 M guanidine X HCl extracts of tissues by radioimmunoassays, further indicating stoichiometric complexes. However, lower levels of nidogen than laminin were found in tissue and cell culture media. A less than 2-fold increase in nidogen was found when F9 cells were stimulated to differentiate with retinoic acid and dibutyryl cAMP, compared to a 30-fold increase in laminin secretion.

Animals↗
PubMed1985

Basement membrane proteins, interstitial collagens, and fibronectin in neurofibroma.

The distribution and nature of extracellular matrix proteins in neurofibroma tissue was studied by indirect immunofluorescence, immunoelectron microscopy, immunoblotting, and rotary shadowing. The most striking feature was an extensive network of basement membranes localized mainly around Schwann cells and small blood vessels. The major components, collagen IV, laminin, and nidogen, were mainly deposited in the lamina densa. Some laminin and nidogen could be extracted with 0.5 M NaCl and were shown by electrophoresis to have the characteristic chain and fragment patterns described previously for these proteins isolated from the mouse Engelbreth-Holm-Swarm (EHS) sarcoma. Fragments of collagen IV and collagen VI were solubilized by limited proteolytic digestion and identified after rotary shadowing. The more remote interstitial regions of the tumor contained cross-striated collagen fibrils which were composed of collagen III (diameter, 20-30 nm) or collagen I (diameter, 40-50 nm). Collagen fibrils thicker than 80 nm were not found. The interstitial regions also contained collagen VI as a fine filamentous network near cells and between collagen fibrils. Deposits of fibronectin were rather small and showed a scattered distribution. The data indicate that Schwann cells contribute considerably to matrix production in neurofibroma which may therefore be a suitable model for studying basement membranes of neuroectodermal origin.

Basement Membrane↗
PubMed1985

Structure and macromolecular organization of type VI collagen.

Collagen VI is a large, disulfide-bonded protein complex which is widely distributed in connective tissue. The constituent polypeptide chains (Mr = 110,000-140,000) consist of collagenous and noncollagenous segments, are degraded to chains of about half the size when collagen VI is solubilized by pepsin, and assemble to a unique pattern of oligomers. As revealed by electron microscopy, the triple-stranded protomer consists of a triple helix 105 nm in length flanked on each side by globular domains of similar size (diameter about 7 nm). Protomers are assembled to dimers by an antiparallel staggered alignment of triple-helical segments. This leads to inner regions, 75 nm in length, of two slightly supercoiled triple helices flanked by globular domains. At both sides 30-nm-long outer triple-helical segments emerge that are terminated by globules. Tetramers are formed from laterally aligned dimers that cross with their outer triple-helical segments in a scissors-like fashion. The same structures, except with much smaller globular domains, are found in pepsin-treated collagen VI. Disulfide-linked collagen VI produced by cultured fibroblasts has a size similar to that of genuine collagen VI found in tissue extracts. Larger forms of collagen VI are assembled from tetramers by end-to-end aggregation which because of an overlap of the outer segments brings all globular domains close together. This arrangement predicts microfibrillar structures in tissues with a periodicity of 100-110 nm and a diameter of 5-10 nm. Structures consistent with this proposal were indeed found by immunoelectron microscopy of placenta and aorta using the ferritin technique. Large, lateral aggregates of collagen VI microfibrils may in addition exist in cell cultures and tissues ("zebra collagen," "Luse bodies") and are presumably maintained by contacts between globular domains.

Animals↗
PubMed1985

Structure and biology of the globular domain of basement membrane type IV collagen.

A procedure was developed for purifying the globular domain NC1 of basement membrane collagen from collagenase digests of a variety of tissues. The globule (Mr = 170,000) is a hexameric structure originating from two collagen IV molecules that are cross-linked at their COOH-terminal ends. Dissociation into subunits derived from alpha 1(IV) and alpha 2(IV) chains occurs at a pH below 4 and after denaturation (8 M urea). The subunits obtained include monomers (Mr = 28,000) and two different dimers (Da,Db) which are connected by disulfide bonds (Db) and/or nonreducible bonds (Da). Almost perfect reconstitution to hexamers is obtained in neutral buffer with mixtures of the subunits or purified dimers but not with purified monomers. Stabilization by dimer formation and other physical data suggest conformationally distinct segments within the subunits, which is also supported by a repeating subdomain structure deduced from cDNA sequences. Monocline crystals of NC1 give a sufficiently detailed X-ray diffraction pattern that should permit elucidation of the three-dimensional structure of the hexamer. Antibodies raised against the globular domain react with all subunits and mainly recognize epitopes stabilized by internal disulfide bridges and/or the hexameric assembly. Immunoprecipitation tests with these antibodies demonstrated a slightly larger subunit size of NC1 in PYS-2 cell culture and the rapid release of precursor-specific segments prior to secretion from the cells. Autoantibodies against mouse tumor NC1 were produced in mice and were detected both in the blood and as tissue-bound forms (kidney, lung). The autoantibody response is accompanied by certain pathological alterations mimicking Goodpasture's syndrome. The possible relationship between the two diseases is substantiated by reaction of Goodpasture antisera with the globular domain obtained from various tissue sources.

Animals↗