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Biomedical subjects

U Hahn

Publications and source records attributed to U Hahn.

At least 181 records · Page 10Linked to original sources
PubMed1989

Binding of vanadate (V) to ribonuclease-T1 and inosine, investigated by 51V NMR spectroscopy.

Ribonuclease T1 (RNase-T1) from Aspergillus Oryzae cleaves ribonucleic acid specifically at guanosine to yield oligonucleotides with terminal guanosine-3'-phosphate. It forms a complex with vanadate (association constant K approximately 145 +/- 30 M-1; delta (51V) = -514 ppm) with spectral features similar to the less stable complexes obtained with di- and tripeptides (Gly-His, Pros-His-Ala, Gly-His-Lys, Val-Glu) containing amino acids that are constituents at the active site of the enzyme. Guanosine also forms a (sparingly soluble) complex with vanadate. Its role is mimicked by inosine, which yields two soluble complexes with vanadate, characterized by delta values of -511 (K = 94 M-1) and -523 ppm (K = 305 M-1 in TRIS buffer and 685 m-1 in buffer-free solution). Comparison with literature values leads to an assignment of the delta = -523 signal to a complex where monovanadate, possibly in a trigonal bipyramidal geometry suggested for the transition state of the phosphate analogue, is coordinated to the 2'- and 3'-oxygens of the ribose ring. A drastic increase of complex stability is observed in the ternary vanadate (12-16 mM)/inosine(10.5 mM)/RNase-T1(5.4 mM) system. An approximate lower limit for the association constant is 1.5.10(5) M-2. The spectral characteristics of the main component of the binary vanadate/inosine complex are essentially maintained (delta = -525 ppm, half-width = 960 Hz), suggesting vanadate binding to the enzyme through hydrogen bonds.

Endoribonucleases↗
PubMed1989

Differentiation of rat intestinal epithelial cells is induced by organotypic mesenchymal cells in vitro.

Stromal-epithelial interaction is a potent driving force in the developing intestinal mucosa which ensures tissue specific cellular differentiation. The mechanisms involved are relevant to tissue renewal in adult organs yet they have not been elucidated because of the lack of appropriate in vitro models. In this study, we have investigated the interaction between intestinal mesenchymal and epithelial cells at the cellular level in vitro. Fetal rat intestinal epithelial cell colonies explanted in vitro on the 15th day of gestation, which failed to mature in plain monocultures, were reassociated in coculture with three different types of mesenchyme:fetal skin, gastric and intestinal mesenchyme. Only fetal epithelial cells cocultured with intestinal (homologous) mesenchyme acquired definite signs of differentiation within three to six days. These primitive epithelial cells were shown by electronmicroscopy to become highly polarized, connected by tight junctions and covered with a regular brush border. Three brush border enzymes were strongly expressed in homologous cocultures and their activity was sensitive to dexamethasone. In contrast, fetal epithelial cells cocultured with skin or stomach derived mesenchyme under identical conditions failed to differentiate in vitro: they remained flat, unpolarised and expressed only low enzyme activity. The unique potential of the small intestinal mesenchyme to promote intestinal epithelial differentiation is discussed.

Animals↗
PubMed1989

[Endemic sprue].

Coeliac disease or Gluten enteropathy is a well-defined, but in its pathogenesis poorly understood syndrome. Diarrhoea, steatoroea and malnutrition due to damage and transformation of the small intestinal mucosa are induced by cereal proteins (gliadin) in genetically predisposed children and adults. Local interactions between immunocompetent cells and structural elements of the small intestinal mucosa have been investigated by immunochemical, ultrastructural and cell biological methods. In this review we discuss the events produced by defined gliadin derivates in vivo and in vitro. They suggest a complex mucosal reaction pattern, involving inflammatory and hyperergic manifestations. The cytotoxicity of gliadin is discussed in the context of defined mediators of lymphocyte and inflammatory cell interactions. The importance of structural elements of the lamina propria and the epithelium as target tissues of the immunological attack and its impact on the local environment is elucidated. Gliadin hypersensitivity is regarded as a genetically determined disposition which can be correlated to the HLA system. The distribution of HLA class I and II antigens with respect to T- and B-lymphocyte functions is described. The diagnosis of coeliac disease and the scientific exploration of cell culture techniques have been greatly improved by modern endoscopy. However, the specificity and sensitivity of laboratory tests for coeliac disease are still controversial The value of determining autoantibodies and antigliadin antibodies is evaluated. Clinical syndromes associated with coeliac disease typically involve immunological phenomena and a comprehensive review of the relevant experiences will be presented. Finally, the general prognosis of coeliac disease and the risk of developing malignancies with respect to the necessity and feasibility of a stringent, lifelong gluten-free diet and patient compliance are discussed.

Celiac Disease↗
PubMed1988

Cloning of a full-length complementary DNA for fatty-acid-binding protein from bovine heart.

A full-length cDNA for bovine heart fatty-acid-binding protein (H-FABP) was cloned from a lambda gt11 cDNA library established from bovine heart muscle. The cDNA sequence shows an open reading frame coding for a protein with 133 amino acids. Colinearity with the amino acid sequences of four tryptic peptides was asserted. H-FABP isolated from bovine heart begins with an N-acetylated valine residue, however, as derived from analysis of the tryptic, amino-terminal-blocked peptide and the molecular mass of the peptide obtained via secondary-ion mass spectrometry. The molecular mass of the total protein is 14673 Da. Bovine H-FABP is 89% homologous to rat H-FABP and 97% homologous to the bovine mammary-derived growth-inhibition factor described recently by Böhmer et al. [J. Biol. Chem. 262, 15137-15143 (1987)]. Significant homologies were also found with bovine myelin protein P2 and murine adipocyte protein p422. Secondary-structure predictions were proposed for these proteins, based on computer analysis, which reveal striking similarities.

Acetylation↗
PubMed1988

Expression of the chemically synthesized gene for ribonuclease T1 in Escherichia coli using a secretion cloning vector.

The gene for ribonuclease T1 from Aspergillus oryzae has been chemically synthesized using the segmental support technique. An Escherichia coli clone producing the ribonuclease at high levels was constructed by linking the gene downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN-III-ompA2. This strategy was employed in order to circumvent a possible toxic effect of the gene product on the host cell. Active ribonuclease containing four additional amino acids at the N-terminus could be isolated from the periplasmic fraction of the host. The final yield after purification was 20 mg enzyme/l liquid culture. With respect to immunological, catalytic and specific behaviour, no qualitative differences could be detected between the enzyme from the over-producing E. coli strain and ribonuclease T1 isolated from A. oryzae.

Aspergillus↗
PubMed1988

Extracellular matrix proteins in small-intestinal cell cultures.

Intestinal cell cultures offer unique possibilities to study the effect of extracellular matrix components on epithelial proliferation and differentiation. We have investigated the specific affinity for distinct matrix proteins, including laminin, collagen types I, III, and IV, and fibronectin, versus neutral control proteins in various small-intestine epithelial cell cultures. Both primary cells and intestinal epithelial cell lines display enhanced affinity for basement membrane constituents compared with interstitial collagens. Only the very undifferentiated, proliferative intestinal epithelial cells also synthesize these proteins, as determined by immunofluorescence and radioimmunoassay. On polarization and maturation, biosynthesis of basement membrane proteins is markedly reduced. Differentiation of intestinal epithelial cells is promoted only by laminin. Fibronectin and collagen type IV have no effect. Putative cell membrane receptors for individual basement membrane proteins are discussed.

Animals↗
PubMed1987

Protein dynamics. A time-resolved fluorescence, energetic and molecular dynamics study of ribonuclease T1.

Studies using time-resolved fluorescence depolarization were performed on the internal motion of Trp 59 of ribonuclease T1 (EC 3.1.27.3) in the free enzyme, 2'-GMP-enzyme complex and 3'-GMP-enzyme complex. The Trp 59 motion was also studied in the free enzyme using molecular dynamics simulations. Energetic analysis of activation barriers to the Trp 59 motion was performed using both the transition state theory and Kramers' theory. The activation parameters showed a dependence on solvent viscosity indicating the transition state approach in aqueous solution to be inadequate. When taking solvent viscosity contributions into account agreement between the transition state and Kramers' theories was obtained. The results indicate the three enzyme forms to have different conformations with the free enzyme and 3'-GMP-enzyme complex being similar. Comparison of the experimental and theoretical results showed a good agreement on the Trp 59 motion in the free enzyme. Trp 59 appears to vibrate rapidly, with a relaxation time of the order of 1 ps, within free space in the protein matrix and to have a slower motion, with a relaxation time of the order of 100 ps, which is related to breathing of the surrounding protein matrix. Molecular dynamics results indicate high mobility in regions of the enzyme involved in the interaction with the guanine base of the inhibitor or substrate while much lower mobility occurred in residues involved in the catalytic mechanism of ribonuclease T1.

Aspergillus oryzae↗
PubMed1987

Intestinal cells produce basement membrane proteins in vitro.

The epithelial-mesenchymal interface of the intestinal mucosa obviously plays an important role in supporting the mucosal architecture. Its significance for the process of migration and differentiation of the epithelial cells remains to be resolved. It consists of a basement membrane, the anchoring zone and the subepithelial connective tissue, the origin of which is unknown. We therefore established an in vitro model to study the development of the endodermal-mesenchymal interface of the fetal human and murine intestinal mucosa. The distribution of the interstitial collagens type I, III, VI and procollagen type III as well as the basement membrane components collagen type IV and laminin was investigated immunohistochemically in these fetal explant cultures. The cultures were also adapted to serum free culture conditions. It was evident that while laminin and collagen type IV could be detected in the primary intestinal epithelium, the formation of an authentic basement membrane required the presence of both the epithelial and the mesenchymal cells. Interstitial collagens and procollagen type III were produced exclusively by the mesenchymal cells. Basement membrane formation in vitro coincided with cytodifferentiation of the endodermal cells as betrayed by electron microscopy and the activity of brush border enzymes. In conclusion, the maturation of the endoderm and the formation of the subepithelial basement membrane require the intimate proximity of viable mesenchyme in vitro.

Animals↗
PubMed1987

Intestinal epithelial cells preferentially attach to a biomatrix derived from human intestinal mucosa.

Primary intestinal epithelial cells have a very short lifespan in vitro when cultured free of mucosal elements. Support of the basal plasma membrane by a more natural substrate may thus enhance the initiation of primary cell cultures. A cell free biomatrix consisting of native interstitial collagens, basement membrane fragments and microfibrils was extracted from the lamina propria of human intestinal mucosa. Immunofluorescence revealed the presence of collagens type III, IV, and VI and procollagens type I and III as well as fibronectin, laminin and undulin. Primary crypt cells of suckling mice displayed a significantly increased affinity to pepsin and collagenase solubilised intestinal biomatrix when compared with plastic and fibronectin. Colonies of primary crypt cells survived for up to four days and longer on pepsin solubilised biomatrix but only for 48 hours on fibronectin. The intestinal biomatrix preparation has proved to be a useful substrate for the initiation and prolongation of primary intestinal cell cultures.

Adult↗
PubMed1985

Crystallization of the activated ternary complex of ribulose-1,5-bisphosphate carboxylase-oxygenase isolated from Rhodospirillum rubrum and from an Escherichia coli clone.

Ribulose-1,5-bisphosphate carboxylase-oxygenase was purified from the photosynthetic bacterium Rhodospirillum rubrum as well as from an Escherichia coli clone overproducing the enzyme. Although the latter enzyme contains 25 additional amino acid residues at the N terminus, both preparations yielded isomorphous tetragonal, bipyramidal crystals of the ternary complex of the enzyme with CO2 and Mg2+. Crystallization is sensitive to variation in pH and to the addition of the transition state analog, 2-carboxyarabinitol-1,5-bisphosphate. The systematic absences in the X-ray diffraction photographs suggest a tetragonal space group P4(3)2(1)2 or the enantiomorph P4(1)2(1)2 with cell dimensions a = b = 83 A, c = 290 A. There is one molecule per asymmetric unit. The resolution on still photographs is 3 A. The crystals are comparable to some of those already published but differ from others.

Crystallography↗
PubMed1985

Two-dimensional 1H NMR investigation of ribonuclease A and ribonuclease-A--pyrimidine-nucleotide complexes.

Ribonuclease A was studied by two-dimensional 1H NMR spectroscopy. 10 out of 12 alanine and 9 out of 10 threonine spin systems as well as all valine [9], leucine [2] and isoleucine [3] spin systems were identified from the correlated spectroscopy (COSY) and relayed coherence transfer spectroscopy (RCT). Sequence-specific assignments were obtained from nuclear Overhauser effect spectra for proton resonances of 21 amino acid moieties. 2' and 3'-pyrimidine-nucleotide-RNase-A complexes were also investigated by two-dimensional NMR. We were able to monitor structural changes in the active center, the vicinity of the active center and in regions far from the catalytic region. Chemical shift changes of resonances of protons near Thr-45 reflected the binding of the same moiety. This in turn is also dependent on the position of the nucleotide phosphate group. Binding of 2' nucleotides led to characteristic changes in protein regions not affected by the binding of 3' nucleotides. These results are interpreted in terms of structural differences between the 2' and 3'-nucleotide-RNase-A complexes; the structure of the complex of the native 3' nucleotide inhibitor being more closely related to that of the free protein.

Amino Acid Sequence↗
PubMed1985

Single and twinned crystals of ribulose-1,5-bisphosphate carboxylase-oxygenase from Alcaligenes eutrophus.

Ribulose-1,5-bisphosphate carboxylase-oxygenase (L8S8) from Alcaligenes eutrophus has been crystallized by equilibrium vapor diffusion techniques with ammonium sulfate as precipitant. Crystals thus obtained either as the ternary complex with CO2 and Mg2+ or as the quaternary complex with CO2, Mg2+, and 2-carboxyarabinitol 1,5-bisphosphate, a transition state analogue, diffract at least to 2.8-A resolution. Both are essentially isomorphous to each other, having orthorhombic space group C222(1) with cell dimensions a = 159 A, b = 159 A, and c = 200 A, and there is half a molecule in the asymmetric unit. The crystals of the ternary complex are sometimes twinned about the c axis so that the space group appears to be tetragonal. In this light, our earlier report (Bowien, B., Mayer, F., Spiess, E., Pähler, A., Englisch, U., and Saenger, W. (1982) Eur. J. Biochem. 106, 405-410) on a tetragonal space group P4(2)2(1)2 with crystals obtained from the same enzyme with Mg2+ and CO2 but without 2-carboxyarabinitol 1,5-bisphosphate might be incorrect.

Alcaligenes↗
PubMed1985

1H and 15N NMR investigation of the interaction of pyrimidine nucleotides with ribonuclease A.

Extensive 1H and 15H NMR investigations of the nucleotide moieties capable of hydrogen bonding to ribonuclease A were carried out in order to gain more detailed information on the specificity of nucleotide-enzyme interaction. The 1H investigations focussed on those protons presumed to be involved in hydrogen bonding between the various nucleotides and the enzyme. In particular these were the imino protons of the uridine nucleotides and the amino protons of the cytidine nucleotides. The technique of 15N-1H double quantum filtering was applied for observation of the resonances of the latter in the nucleotide-enzyme complex. The downfield shift observed for the imino proton resonance of the uridine nucleotides was indicative of hydrogen bond formation to the enzyme. 15N NMR spectra of the free nucleotides and the nucleotide-enzyme complexes were also acquired to examine the possibility of hydrogen bond formation at the N3 site of both pyrimidine bases and the amino group of the cytidine nucleotides. The downfield shift observed for the 15N3 resonance of the uridine nucleotides and the upfield shift observed for the corresponding resonance of the cytidine nucleotides was evidence that the N3 moiety acts as hydrogen donor or hydrogen acceptor in the nucleotide-enzyme complex. The effect of complex formation on the 15N1 resonance of the respective bases was also studied. Both 1H and 15N NMR results indicated subtle differences between the complexes of the 2' and 3' nucleotides. The extent of hydrogen bonding as well as the arrangement of the nucleotide base at the active site of the enzyme varies in dependence on the position of the phosphate group. It is established that hydrogen bonding, though not the main binding force between the nucleotides and the enzyme, is certainly a major factor of RNase A specificity for pyrimidine nucleotides.

Chemical Phenomena↗
PubMed1985

Cytotoxicity of lectins on rat intestinal mucosa enhanced by neuraminidase.

The lectins wheat germ agglutinin (WGA) and Concanavalin A (ConA) were perfused into an isolated small intestinal segment alone or after prior perfusion with neuraminidase for a 10 day period in the rat. Intestinal morphometry, intraepithelial Lymphocyte (IEL) and round cell content as well as digestive capacity was measured in the loop and in the adjacent segments. Both lectins induce a mucosal transformation in all segments but ConA is more effective than WGA. Pre-incubation with neuraminidase enhances the action of ConA throughout, whereas only a partial enhancement of the effect of WGA is observed. The mucosal transformation after long term perfusion with the lectins resembles the hyperregenerative adaptation of the mucosa due to gluten in coeliac disease and may thus serve as an animal model for this disease.

Animals↗
PubMed1983

Consensus structure and evolution of 5S rRNA.

A consensus structure model of 5S rRNA presenting all conserved nucleotides in fixed positions has been deduced from the primary and secondary structure of 71 eubacterial, archaebacterial, eukaryotic cytosolic and organellar molecules. Phylogenetically related groups of molecules are characterized by nucleotide deletions in helices III, IV and V, and by potential base pair interactions in helix IV. The group-specific deletions are correlated with the early branching pattern of a dendrogram calculated from nucleotide substitution data: the first major division separates the group of eubacterial and organellar molecules from a second group containing the common ancestors of archaebacterial and eukaryotic/cytosolic molecules. The earliest diverging branch of the eubacterial/organellar group includes molecules from Thermus thermophilus, T. aquaticus, Rhodospirillum rubrum, Paracoccus denitrificans and wheat mitochondria.

Bacteria↗