Search PubMed⌕ Search

Biomedical subjects

K Mann

Publications and source records attributed to K Mann.

At least 379 records · Page 21Linked to original sources
PubMed1991

Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV.

Basement membrane protein BM-40, prepared from the mouse Engelbreth-Holm-Swarm tumor, was used in native, denatured and proteolytically processed form for binding to various extracellular matrix proteins. BM-40 and its derivatives were also characterized by CD spectroscopy, calcium binding and epitope analysis. Of several basement membrane proteins tested only collagen IV showed a distinct and calcium-dependent binding of BM-40 in an immobilized ligand assay. This interaction was specific as shown by a low activity of other collagen types (I, III, V, VI) in direct binding and competition assays. The binding was reduced or abolished by metal-ion-chelating or chaotropic agents, high salt and reduction of disulfide bonds in BM-40. Fragment studies indicated that domains III (alpha-helix) and/or IV (EF hand) of BM-40 possess the binding site(s) for collagen IV, while the N-terminal domains I and II provide the major antigenic determinants. A major BM-40-binding site on collagen IV was dependent on a triple-helical conformation and could be localized to a pepsin fragment from the central portion of the triple-helical domain, in agreement with electron microscopic visualization of BM-40--collagen-IV complexes.

Amino Acid Sequence↗
PubMed1991

Class pi glutathione S-transferase from pig lung. Purification, biochemical characterization, primary structure and crystallization.

A cytosolic glutathione S-transferase from pig lung was purified 210-fold to apparent homogeneity. The enzyme was classified as a class pi isoenzyme on the basis of its physical and chemical properties. It is homodimeric with a subunit Mr of 23,500, has a pI of 7.2, and shows a high specific activity towards ethacrynic acid. The glutathione analogues, S-hexylglutathione and glutathione sulfonate, were strong reversible inhibitors. The enzyme's primary structure, established entirely by protein chemical methods, consists of 203 amino acids and is highly similar (82-84% residue identity) to the rat and human class pi isoenzymes. Furthermore, there was no evidence of microheterogeneity or post-translational modifications. Each subunit contains a highly reactive cysteine residue, the modification of which leads to enzyme inactivation. None of the cysteine residues in the pig enzyme appear to form intramolecular disulfide bonds. Singel crystals of the glutathione-S-transferase-glutathione-sulfonate complex were obtained by the hanging-drop method of vapour diffusion from poly(ethylene glycol) 4000 solutions. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of a = 10.125 nm, b = 8.253 nm and c = 5.428 nm and diffract to better than 0.22 nm.

Amino Acid Sequence↗
PubMed1991

Biosynthesis and secretion of an osteopontin-related 20-kDa polypeptide in the Madin-Darby canine kidney cell line.

We describe a 20-kDa phosphorylated polypeptide, which is secreted constitutively at the apical surface of the kidney-derived Madin-Darby canine kidney cell line. Using polyclonal antibodies raised against this protein, we show that it is generated from a 60-kDa O-glycosylated, sulfated, and phosphorylated precursor protein by an intracellular proteolytic maturation step, which is pH-sensitive. Amino acid sequence analysis of the 20-kDa secreted polypeptide demonstrated that it displays 70% identity with the carboxyl-terminal amino acids of human osteopontin. The amino-terminal amino acid of the 20-kDa polypeptide corresponds to amino acid 213 of human osteopontin. Thrombin has been shown to cleave rat osteopontin in vivo and in vitro at amino acid 153, yielding two fragments of 28 and 26 kDa. A similar cleavage product can be detected by thrombin treatment of the 60-kDa precursor, suggesting that the precursor is identical or closely related to osteopontin. In the rat nephron, the protein has been localized along the luminal surfaces of the proximal and distal tubule and the collecting duct cells. These results show that in the kidney-derived cell line Madin-Darby canine kidney osteopontin or a closely related protein is proteolytically processed to a 20-kDa polypeptide, raising the possibility that diverse functions of osteopontin in various tissues might be attributed to specific processing to distinct polypeptides.

Amino Acid Sequence↗
PubMed1991

Changes of ionic channel distribution in myelinated nerve fibres from rats with experimental allergic neuritis.

Voltage clamp experiments were performed in single myelinated nerve fibres of rats with experimental allergic neuritis (EAN). Nerve fibres from diseased animals showed a varying degree of demyelination. In fibres with extensive demyelination large K+ currents were measured. Analysis of the contribution of slow and fast K+ channels revealed that in these fibres the relative contribution of slow K+ channels was about 50% compared to about 25% in fibres acutely demyelinated as previously reported. This indicates that chronic demyelination changes the distinct distribution of ionic channels in the rat node of Ranvier.

Animals↗
PubMed1991

Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.

Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd-I (positions 1-905) comprising the N-terminal globule and rod-like domain and Nd-II corresponding mainly to the C-terminal globule (position 906-1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N-terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N-terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31-56 kDa) connected by either a rod-like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd-I binding to collagen IV and fragment Nd-II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.

Animals↗
PubMed1991

An abundant chick gizzard integrin is the avian alpha 1 beta 1 integrin heterodimer and functions as a divalent cation-dependent collagen IV receptor.

The alpha-subunit of an abundant chick gizzard integrin was isolated (T. Kelly, L. Molony, and K. Burridge, 1987, J. Biol. Chem. 262, 17,189-17,199) and fragmented by proteolytic digestion. The N-terminal sequences of the intact polypeptide and of several internal peptides were determined and were found to be highly homologous to the mammalian integrin alpha 1-subunit. Monoclonal antibodies to the chick integrin beta 1-chain react on immunoblots with the gizzard integrin beta-subunit (U. Hofer, J. Syfrig, and R. Chiquet-Ehrismann, 1990, J. Biol. Chem. 265, 14,561-14,565). The chain composition of the abundant chick gizzard integrin is therefore alpha 1 beta 1. Polyclonal antibodies to the avian integrin alpha 1-subunit block attachment of embryonic gizzard cells to human and chick collagen IV completely and inhibit attachment to mouse Engelbreth-Holm-Swarm (EHS) tumor laminin partially. In ELISA-style receptor assays, the isolated alpha 1 beta 1 integrin bound to human and chick collagen IV and to mouse EHS tumor and chick heart laminin. While the binding to collagen IV was abolished by removal of divalent cations, the binding to laminin was not sensitive to EDTA under the conditions used. Collagen I bound the isolated avian alpha 1 beta 1 integrin only weakly. As collagen IV was the only extracellular matrix protein for which a consistent, divalent cation-dependent, binding to the avian alpha 1 beta 1 integrin could be demonstrated in both cellular and molecular assays we suggest that it is a preferred ligand for this integrin.

Amino Acid Sequence↗
PubMed1991

Association of topoisomerases I and II with the chromatin in SV40-infected monkey cells.

The subcellular localization of topoisomerase I and topoisomerase II has been compared in Simian virus (SV40)-infected and uninfected TC7 monkey cells. In SV40-infected cells, both of these enzymes are preferentially associated with the chromatin. Some topoisomerase I is associated with the nuclear matrix, whereas topoisomerase II shows no such association. In uninfected TC7 cells, topoisomerase I is present in both the chromatin and nuclear matrix fractions. Topoisomerase II, on the other hand, is not detected in any of the subcellular fractions of uninfected cells. After SV40 infection, there is a marked increase in the level of chromatin-associated topoisomerase II.

Animals↗
PubMed1991

Characterization of a type IV collagen major cell binding site with affinity to the alpha 1 beta 1 and the alpha 2 beta 1 integrins.

The aim of this investigation was to identify the domains of type IV collagen participating in cell binding and the cell surface receptor involved. A major cell binding site was found in the trimeric cyanogen bromide-derived fragment CB3, located 100 nm away from the NH2 terminus of the molecule, in which the triple-helical conformation is stabilized by interchain disulfide bridges. Cell attachment assays with type IV collagen and CB3 revealed comparable cell binding activities. Antibodies against CB3 inhibited attachment on fragment CB3 completely and on type IV collagen to 80%. The ability to bind cells was strictly conformation dependent. Four trypsin derived fragments of CB3 allowed a closer investigation of the binding site. The smallest, fully active triple-helical fragment was (150)3-amino acid residues long. It contained segments of 27 and 37 residues, respectively, at the NH2 and COOH terminus, which proved to be essential for cell binding. By affinity chromatography on Sepharose-immobilized CB3, two receptor molecules of the integrin family, alpha 1 beta 1 and alpha 2 beta 1, were isolated. Their subunits were identified by sequencing the NH2 termini or by immunoblotting. The availability of fragment CB3 will allow for a more in-depth study of the molecular interaction of a short, well defined triple-helical ligand with collagen receptors alpha 1 beta 1 and alpha 2 beta 1.

Amino Acid Sequence↗
PubMed1991

Urinary dolichol--a doubtful marker of alcoholism.

The purpose of this study was to replicate the results of Pullarkat and Raguthu and Roine et al. who found elevated levels of urinary dolichol (long-chain 2,3-dihydropolyprenols) in chronic alcoholic patients. We investigated a sample of 21 alcohol-dependent inpatients voluntarily entering detoxification treatment. Urinary dolichol was only slightly increased as compared to 21 healthy controls. When dolichol was related to urinary creatinine no differences between alcoholic patients and controls could be found. Under conditions of confirmed abstinence the slightly elevated levels of dolichol returned to normal within 2 weeks. Compared with the sensitivity of gamma-glutamyltransferase which ranges from 72-85%, the value of urinary dolichol (sensitivity 9-19%) as a biochemical marker of alcoholism must be doubted.

Adult↗
PubMed1991

Asialoagalacto-human chorionic gonadotropin, a carbohydrate-modified variant of human chorionic gonadotropin, antagonizes the stimulatory actions of bovine thyroid-stimulating hormone on thyroid function and HLA-DR expression in human thyroid in vitro and in vivo.

The concept of using thyroid-stimulating hormone (TSH) receptor antagonists in the management of Graves' disease is intriguing. Therefore, we investigated a TSH receptor antagonist derived from human chorionic gonadotropin (hCG) with respect to TSH receptor binding, adenylate cyclase activity, thyroid hormone release, and HLA class II antigen expression in vitro and in an in vivo model. A variant of hCG, asialoagalacto-hCG, like asialo-hCG and unlike hCG itself, inhibited both 125I-bTSH binding and cAMP response to bTSH in human thyroid membranes. However, like intact or deglycosylated hCG and unlike asialo-hCG, asialoagalacto-hCG displayed a limited affinity for hepatic asialoglycoprotein receptors, a likely marker for its in vivo turnover rate. It proved capable of inhibiting bTSH-stimulated thyroid hormone release in human thyroid slices as well as in the nude mouse bearing human thyroid transplants. It also prevented bTSH induced hypertrophy of transplanted thyrocytes. Further, HLA-DR expression induced by bTSH in the presence of gamma-interferon on human thyrocytes was inhibited. In conclusion, we present evidence that asialogalacto-hCG antagonizes bTSH actions on thyroid function and HLA-DR expression in human thyroid in vitro and, more importantly, in an in vivo model. Hence, the hCG variant described here or similar agents should warrant further exploration in the study and treatment of Graves' disease.

Adenylyl Cyclases↗
PubMed1991

Heterogeneity of autoantibodies against thyroid peroxidase in autoimmune thyroid disease: evidence against antibodies directly inhibiting peroxidase activity as regulatory factors in thyroid hormone metabolism.

The close relationship between thyroid microsomal antigen and thyroid peroxidase (TPO) is now well established. The present study evaluates the significance of autoantibodies against TPO (anti-TPO-Abs) in the various forms and stages of autoimmune thyroid disease with respect to a possible heterogeneous nature and particularly to their influence on TPO activity. When measured by a RIA using purified human TPO, anti-TPO-Abs were highly correlated with microsomal antibodies determined by enyzme-linked immunosorbant assay (r = 0.96; P less than 0.0001) and with the results of a TPO immunoprecipitation assay using crude microsomal preparations (r = 0.76; P less than 0.001). Relating the results of these assays to the reactivities of patients' sera with thyroid microsomes in immunoblot under nonreducing and reducing conditions, discordant results could be observed in a few cases. Further analysis of these data indicate a heterogeneous nature of anti-TPO-Abs, which react with at least two antigenic domains of the TPO molecule. The comparative analysis of patients with hyperthyroid Graves' disease, patients with Graves' disease in clinical remission, and patients with hypothyroid Hashimoto's thyroiditis revealed no significant differences in the antibody spectrum. When evaluating the direct influence of anti-TPO-Abs on the activity of TPO under a rigorous methodological approach, we found no significant inhibition of the enzymatic activity by any of the sera investigated from patients with autoimmune thyroid disease compared to that in sera from normal controls. In conclusion, the data indicate a heterogeneous nature of anti-TPO-Abs. The spectrum of antigenic epitopes recognized by anti-TPO-Abs seems not to be significantly different in the various forms and stages of autoimmune thyroid disease. The lack of an inhibitory effect of autoantibodies on TPO activity argues against direct binding of autoantibodies to the enzymatic sites of TPO and indicates that they are not important factors in producing thyroid dysfunction in autoimmune thyroid disease.

Adult↗
PubMed1991

alpha 2-Macroglobulin is cleaved by HIV-1 protease in the bait region but not in the C-terminal inter-domain region.

alpha 2-Macroglobulin is cleaved by human immunodeficiency virus-1 protease. The cleavage site is the Phe684-Tyr685 bond in the "bait region", an exposed part of alpha 2-macroglobulin, creating the "F-form". The methylamine derivative of alpha 2-macroglobulin is also cleaved at the same bond. The homologous chicken ovomacroglobulin does not form an F-form structure with the protease, although, F-form generation by other enzymes is known. This is possibly due to the lack of a suitable cleavage sequence in the corresponding region of ovomacroglobulin. In human alpha 2-macroglobulin, the interdomain segment between the main part of the molecule and the receptor-binding C-terminal domain is not cleaved by the HIV protease although typical cleavage sequences occur. In AIDS, therefore, HIV protease from infected cells in unlikely to interfere with receptor-binding of alpha 2-macroglobulin.

Amino Acid Sequence↗
PubMed1991

The amino-acid sequence of three proteins of photosystem I of the cyanobacterium Fremyella diplosiphon (Calothrix sp PCC 7601).

Three proteins containing 138 amino acids (psaD protein), 80 amino acids (psaC protein) and 66 amino acids (psaE protein) of the photosystem I (PS I) complex of the cyanobacterium Fremyella diplosiphon (Calothrix sp PCC 7601) were isolated and sequenced. Comparison with previously known sequences showed a close relationship to homologous proteins of Nostoc, another filamentous cyanobacterium.

Amino Acid Sequence↗
PubMed1991

Equine cutaneous amyloidosis derived from an immunoglobulin lambda-light chain. Immunohistochemical, immunochemical and chemical results.

Amyloid deposits from equine cutaneous nodular amyloidosis associated with extramedullary plasmacytoma were classified immunohistochemically as equine immunoglobulin lambda-light chain-derived and designated eA lambda (HIP). For chemical identification, the amyloid fibril proteins were separated on Sephadex G-100 in 6M guanidine.HCl. Polypeptides of predominantly 24 kDa and 50 kDa were found by polyacrylamide gel electrophoresis. They have preponderance of immunoglobulin lambda-antigenic determinants as detected by immunodiffusion and immunoblotting. Since the N-terminus of the major proteins was blocked, peptides were generated with trypsin and endoproteinase Asp-N and then isolated using reversed-phase high-performance liquid chromatography. Automatic amino-acid sequence determination of seven peptides showed novel sequences. Data bank comparison indicated that these peptides were derived from a monoclonal immunoglobulin lambda-light and a gamma-heavy chain. The light chain was considered to be the leading amyloidogenic polypeptide, since it was the predominant component in a virtually pure amyloid fibril preparation. Thus, immunoglobulin lambda-light chain-derived amyloidosis, so far established only in man and cat, has now also been identified in the horse.

Amino Acid Sequence↗
PubMed1991

Course of thyroid iodine concentration during treatment of endemic goitre with iodine and a combination of iodine and levothyroxine.

In the treatment of endemic goitre, the concept of giving levothyroxine in combination with iodine offers a promising therapeutic approach by influencing not only TSH secretion but also intrathyroidal iodine content. However, little is known about the doses of iodine necessary to correct intrathyroidal iodine deficiency. To get more information on this important issue, we conducted a prospective, double-blind study on the effect of a monotherapy with 500 micrograms iodide/day and a combined treatment with 100 micrograms levothyroxine and 100 micrograms iodide/day on thyroid iodine concentration as measured by fluorescence scintigraphy. In a group of 12 patients, a 4-month treatment with 100 micrograms levothyroxine and 100 micrograms iodide/day did not significantly affect thyroid iodine concentration (0.35 +/- 0.14 vs 0.37 +/- 0.11 mg/g). The application of 500 micrograms iodide/day in these patients during a second 4-month period resulted in a sharp increase in thyroid iodine concentration from 0.37 +/- 0.11 to 0.61 +/- 0.14 mg/g (p less than 0.01). Another group of 8 patients first treated with 500 micrograms iodide/day also showed a significant increase in iodine concentration from 0.35 +/- 0.14 to 0.65 +/- 0.20 mg/g (p less than 0.01). After switching to the combination regimen during a second 4-month period, thyroid iodine concentration slightly decreased, particularly in those patients with high iodine concentrations after monotherapy with iodide (0.65 +/- 0.20 vs 0.50 +/- 0.12 mg/g, p less than 0.05). In conclusion, treatment with 500 micrograms iodide/day could sharply increase thyroid iodine concentration in patients with endemic goitre. In contrast, a combination of 100 micrograms levothyroxine and 100 micrograms iodide/day had no significant effect on thyroid iodine concentration.

Adult↗