Now showing items 21-40 of 4236

    • HERSTELLUNG ISOPORER ULTRAFILTRATIONSMEMBRANEN AUS KRISTALLINEN BAKTERIENZELLWANDSCHICHTEN

      Sleytr, U. B.; Sara, M.; Zentrum für Ultrastrukturforschung, Universität für Bodenkultur, Wien (1986)
      Viele Eu- und Archaebakterien besitzen eine kristalline Protein- oder Glykoproteinschicht (S-Schicht) als äußerste Zellgrenzfläche. Über | eine Reihe einfacher Verfahrensschritte ist es möglich, S-Schichten | zu isolieren und an geeignete poröse Träger zu binden. Auf diese Weise kann ein völlig neuartiger Typ von Ultrafiltrationsmembranen hergestellt werden, dessen aktive Trennschicht sich durch absolute Isoporosität auszeichnet und über ein breites Spektrum proteinchemischer Reaktionen spezifisch modifiziert werden kann.
    • OPTIMIERUNG EINES MEMBRANBEGASUNGSSYSTEMS ZUR BLASENFREIEN SAUERSTOFFVERSORGUNG TIERISCHER ZELLKULTUREN

      Kuhlmann, W.; B. Braun Melsungen AG, 3508 Melsungen (1986)
      Bei hoher Zelldichte kann das Wachstum tierischer Zellkulturen in Suspension durch die Versorgung mit Sauerstoff limitiert werden. Während in kleineren Volumina der Sauerstoffeintrag über die Oberfläche in der Regel ausreicht, ist für Suspensionskulturen im größeren Maßstab die gleichzeitige Erfüllung folgender Forderungen notwendig: - Homogene Suspendierung von Zellen oder Microcarriern; - Geringe Scherkräfte beim Rühren und Begasen unter Gewährleistung ausreichender Sauerstoffversorgung auch für Zellen mit hohem 02-Bedarf; - Hohe Mischgeschwindigkeit; - Möglichkeit der Maßstabsvergrößerung; - Möglichkeit der Nachrüstung an herkömmlichen Rührkessel-Fermentern. Am Beispiel des Laborfermenters BIOSTAT M mit 1,5 1 Arbeitsvolumen wird die Entwicklung und Funktion eines Begasungssystems, welches diesen Forderungen entspricht, aufgezeigt.
    • Anwendung von Membranen bei der Aufarbeitung von Interferon-ß

      Menge, Ulrich; GBF, Gesellschaft für Biotechnologische Forschung mbH., Mascheroder Weg 1 D-3300 Braunschweig, FRG (1986)
      Ultrafiltrationen von Interferon-ß ergeben im Unterschied zu solchen von Interferon-a unreproduzierbare und teils sehr hohe Aktivitätsverluste. Die Versuchsergebnisse zeigen, daß diese Verluste zum Teil auf einer Adsorption des Proteins an Membranen beruhen. Diafiltrationen mit einer gleichzeitigen Anderung des pH-Wertes führen zu weiteren hohen, proteinspezifischen Verlusten‘ Solche Verluste können jedoch wegen der hohen Produktionskosten für Interferon- 8 nicht akzeptiert werden und daher kommen Membranprozesse bei der Herstellung pharmazeutischer Präparate von Interferon-ß aus tierischen Zellkulturen nur bedingt in Frage. Da Interferon-8 von einigen Kunststoffen stark, von anderen aber wiederum nicht absorbiert wird, sind neue Membranpolymere von Interesse.
    • MEMBRANPROZESSE BEI DER AUFARBEITUNG BIOLOGISCH AKTIVER PROTEINE

      Kula, Maria-Regina; Kroner, Karl Heinz; GBF, Gesellschaft für Biotechnologische Forschung mbH., Mascheroder Weg 1, D-3300 Braunschweig (1986)
      Es wird eine Übersicht gegeben über Einsatzmöglichkeiten und verfahrenstechnische Aspekte von Membrantrennverfahren: zur Konzentrierung biologisch aktiver Proteine durch Ultrafiltration, Entsalzen durch Diafiltration und Fest-Flüssig-Trennung durch Querstromfiltration an mikroporösen Membranen.
    • ABTRENNUNG VON POLYETHYLENGLYKOL AUS PROTEINHALTIGEN LOSUNGEN MIT CELLULOSE-ACETAT-MEMBRANEN

      Papamichael, Neophytos; Kula, Maria-Regina; GBF, Gesellschaft für Biotechnologische Forschung mbH., Mascheroder Weg 1 D-3300 Braunschweig (1986)
      Polyethylenglykole wurden mit Cellulose-Acetat-Membranen ultrafiltriert, um den Einfluß hydrodynamischer Verhältnisse auf das Retentionsverhalten zu messen. Dabei wurden Lösungen ohne bzw. mit Rinder-Serumalbumin (BSA) verwendet. Die Reduzierung der Grenzschichtdicke an der Membranoberfläche durch Erhöhung der Rührergeschwindigkeit bewirkt eine Steigerung des Fluxes und des Retentionskoeffizienten. Bei zunehmendem Differentialdruck wird die Retention ebenfalls größer. Bei Diafiltrationen von proteinhaltigen Lösungen wurde ein bisher nicht bekanntes Phänomen beobachtet: Nach einer Zunahme der Retention von 30 bis auf 60% (vergl. reine PEG-Lösung) geht das Rückhaltungsvermögen der Membran mit steigender BSAKonzentration zurück. Dieses Verhalten wird in dem Beitrag diskutiert.
    • MIKROFILTRATION VON FERMENTERBRÜHEN

      Rähse, W.; Carduck, F.-J.; HENKEL KGaA, 4000 Düsseldorf (1986)
      In der Biotechnologie stellt die Crossflow-Mikrofiltration ein modernes Verfahren zur Aufarbeitung von Fermenterbrühen dar. Bei intrazellulären Inhaltsstoffen können die Zellen mit Hilfe dieser Technik vor ihrem Aufschluß angereichert werden /1/. Im Falle von extrazellulären Wertstoffen ist es möglich, diese durch die Mikrofiltrationsmembran von den Zellen sowie von den aus dem Nährmedium stammenden Feststoffen abzutrennen. Die Vorgehensweise beider Isolierung gelöster Wertstoffe mittels der Crossflow-Mikrofiltration wird am Beispiel der alkalischen Protease beschrieben, wobei insbesondere die Abhängigkeiten des Permeatflusses und der Retention vom Membranmaterial, von der mittleren Porengröße der Membran, von den Betriebsbedingungen, den physikalischen Daten der Fermenterbrühe und vom Feststoffzusatz diskutiert und quantifiziert werden.
    • INHIBITORS OF OLIGOSACCHARIDE PROCESSING

      Fuhrmann, Ulrike; Bause, Ernst; Ploegh, Hidde; Institute for Genetics and Biochemistry, University of Cologne, Cologne (F.R.G) (1987)
      Most proteins present on the surface of eukaryotic cells and proteins secreted by them are glycoproteins. Glycoproteins, generally speaking, may contain asparagine- -linked (N-linked) or serine/threonine linked (0-linked) oligosaccharides. A given protein may contain either one or both types of oligosaccharides. Notwithstanding their common occurrence -glycoproteins are found in bacteria, yeasts and all higher eukaryotes- there is no consensus on the function of protein bound glycans. They are thought to be involved in protection against proteolysis and contribute to or modify the folding of the polypeptide backbone. Protein bound glycans are also thought to constitute signals for intracellular traffic of glycoproteins, and may participate in determining the specificity of interactions between receptors and ligands and the interactions between cells. In present-day biotechnology, the importance of protein bound carbohydrate is likely to come under close scrutiny. Products produced by genetic engineering are often derived from cells or organisms that do not normally produce these substances (e.g. interferon production in bacteria) yet are intended for use in clinical applications. It is therefore of paramount importance to determine what, if any, role is played by protein bound carbohydrate. Parameters such as half-life in vivo, interaction with receptors, immunogenicity and the like may all be affected. Surprisingly enough, the armamentarium available for modifying carbohydrates in a predictable and directed manner is scarce. Through detailed knowledge of glycan biosynthesis, opportunities may be created to fill this gap. This article deals with inhibitors of N-linked oligosaccharide processing. These substances, most of which were described only recently, show promise of allowing the manipulation of N-linked glycan structures in a rather precise manner, and hence might be useful in investigations on the function of carbohydrates. In order to describe the effects of these inhibitors in their proper context, a4 brief overview of N-linked glycan synthesis will be given. Subsequently, the literature on the enzymes involved in N-linked glycan processing will be reviewed in some detail. Since the effects of the inhibitors to be described need not be the same for each of the enzymes involved even if these enzymes have very similar substrate specificities, their description as possibly distinct targets for inhibition is called for. The inhibitors of oligosaccharide trimming themselves will then be described in detail. Finally, the concluding remarks will deal with some possible applications of N-linked glycan processing inhibitors.
    • SYNTHESES OF COMPLEX OLIGOSACCHARIDE SEQUENCES OF GLYCOCONJUGATES

      Paulsen, Hans; Institut für Organische Chemie der Universität Hamburg, Martin-Luther-King-Platz 6, D-2000 Hamburg 13, Fed. Rep. of Germany (1987)
      The methods of chemically synthesizing complex oligosaccharides have made amazing progress in the past few years. The three basic reactions, especially suited for the selective linking of saccharide units, are characterized. In particular, examples of syntheses, which deal with the construction of oligosaccharide chains from the area of glycolipids and oand N-glycoproteins, are presented. The new methods of conformation analysis are explained, exemplified by an octasaccharide structure. Information concerning the molecular form of an oligosaccharide is given.
    • BIOTINYLATED OLIGONUCLEOTIDE HYBRIDIZATION PROBES: PROGRESS TOWARDS A NON-RADIOACTIVE METHOD FOR THE DIAGNOSIS OF HUMAN GENETIC DISEASES.

      Wallace, R. Bruce; Studencki, Anna B.; Murasugi, Akira; Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, CA. 91010, USA. (1987)
      Under appropriate conditions synthetic oligonucleotide hybridization probes display essentially absolute hybridization specificity. All of the non-Watson-Crick base pairs, including G-T, have a destabilizing effect. Thus, it is possible to choose stringent conditions of hybridization such that, while a perfectly matched duplex between an oligonucleotide and complementary DNA will form, duplexes mismatched at one or more positions will not. The hybridization of oligonucleotides to regions of DNA containing the single base changes responsible for many human genetic disease, thus, provides a means of detecting these diseases. In an attempt to develop a non-radioactive method for the detection of human genetic diseases, we have prepared biotinylated oligonucleotides by an enzymatic method. An oligonucleotide probe (23-mer) containing a single biotinylated deoxyuridine residue at the 3’-terminus was prepared by a primer extention reaction using E. coli DNA polymerase I (Klenow fragment). The biotinylated probe could be hybridized to a complementary sequence and visualized by Avidin D and biotinylated alkaline phosphatase. A general approach to the enzymatic synthesis of oligonucleotides containing a single biotinylated deoxyuridine at the 3’ end is described.
    • A NEW APPROACH TO THE SYNTHESIS OF GLYCOCONJUGATES

      Schmidt, Richard R.; Fakultät für Chemie, Universität Konstanz D-7750 Konstanz, Germany (1987)
      1-0-Unsubstituted aldoses afford with trichloroacetonitrile under base catalysis 0-(glycosyl)trichloroacetimidates in high yields. Depending on the reaction conditions either 0-(a-glycosyl) or 0-(ßB-glycosyl)trichloroacetimidates may be obtained highly selectively or even exclusively. The extraordinary diastereoselectivity control is mainly due to stereoelectronic effects. The 0-(a- or B-glycosyl)trichloroacetimidates are stable compounds, which can be isolated easily, quite often as erystalline material. They may be stored for a long period of time. However, upon mild acid catalysis they become strong glycosylating agents. They have proven as versatile intermediates in complex glycosidation reactions with different nucleophiles (0-, S-, N-, and C-nucleophiles). The application of this methodology to the synthesis of glycosphingolipids, glycophospholipid type compounds, and to the synthesis of the tetrasaccharide of the core region of 0-glycoproteins is presented. In addition, the 0-(glycosyl)trichloroacetimidate based synthesis of the repeating unit of Neisseria meningitidis (Serogroup L) will be outlined. Halogenoses and heavy metal salt catalysis are not required in these procedures.
    • CHEMICAL SYNTHESIS AND BIOLOGICAL APPLICATIONS OF OL IGODEOXYNUCLEOTIDES CONTAINING CARCINOGEN-DNA ADDUCTS: O6-METHYLGUANINE AND N-(GUAN-8-YL)- 4-AMINOBIPHENYL

      Lasko, D. D.; Fowler, K. W.; Green, C. L.; Loechler, E. L.; Weis, C. C.; De Rosa, L. M.; Kadlubar, F. F.; Essigmann, J. M.; Laboratory of Toxicology Department of Nutrition and Food Science and lDepartment of Chemistry Massachusetts Institute of Technology, Cambridge, MA 02139 USA; National Center for Toxicological Research Jefferson, AR 72079 USA (1987)
      Tetranucleotides containing the carcinogen-DNA adducts 06-methylguanine and N-(guan-8-yl )-4-aminobiphenyl were synthesized and the structures of these adducted oligomers were characterized. Both tetramers were efficiently incorporated into E. coli bacteriophage M13 DNA containing a four-base gap in the unique recognition site for the restriction endonuclease PstI. Each lesion inhibited cleavage of the respective adducted genome by PstI. After introduction into E. coli, 06-methylguanine induced mutations at the PstI site. In cells with normal 06-methylguanine DNA repair capability, the frequency of mutant phage was 0.4%. When cells were depleted in their ability to repair this lesion, the mutation frequency increased. The highest mutation frequency attained was 20%. In both repair competent and repair depleted cells, the mutants induced by 06-methylguanine were exclusively G to A transitions.
    • BASIC SUGAR DERIVATIVES, TOOLS FOR MAPPING THE ACTIVE SITE OF GLYCOSIDASES AND FOR THE STUDY OF GLYCOPROTEIN BIOSYNTHESIS

      Legler, Günter; Institut für Biochemie, Universität Köln Zülpicher Str. 47, D-5000 Köln 1 (1987)
      Most glycoside hydrolases are strongly inhibited by sugar derivatives with a basic nitrogen adjacent to their C-1. This is assumed to be due to the formation of an ion pair at the catalytic site consisting of the protonated inhibitor and a negatively charged group essential for catalysis. While glycosylamines and their N-alkylderivatives are useful for kinetic studies to characterize the active site with respect to charge distribution, hydrophobic properties and solvent accessibility,their life-time in aqueous solution is too short for long term studies. Stable sugar derivatives with suitable properties are 5-amino-5-deoxyhexopyranoses and 1,5-dideoxy-1,5-imino hexitols. The basic principle of their synthesis is the oxidation of a protected furanoid monosaccharide at C-5 and conversion of the resulting ketone to the epimeric amines by reduction of its oxime. Deprotection provides the pyranoid sugar derivative with the basic nitrogen in the ring. In glycoprotein biochemistry these inhibitors have been used to specifically inhibit the 'trimming' glycosidases that catalyze essential steps in the conversion of the primary asparagine linked Gle Man (GlcNAc) unit to complex type or high mannose type glycans. Interference with these steps is expected to provide insight into the role of glycan structure in surface expression, secretion and receptor binding. Another application is the construction of affinity ligands for the purification of these enzymes.
    • BUILDING BLOCKS FOR THE CHEMICAL SYNTHESIS OF DNA CONTAINING C(3')-CH2-P BONDS

      Morr, Michael; Ernst, Ludger; Kakoschke, Christel; GBF, Gesellschaft fiir Biotechnologische Forschung mbH., Mascheroder Weg 1 D-3300 Braunschweig, F.R.G. (1987)
      The preparation, starting from glucose, of 3'-[ (2-chlorophenoxy)phosphinylmethyl]- N2-isobutyryl-5'-0-(4-methoxytrityl)-2' ,3'-dideoxyguanosine ts described. Applying established phosphotriester methods, 21 was used to synthesize a phosphonate analogue of the dinucleotide monophosphate d(G-C).
    • TOWARDS CONFORMATIONAL SEQUENCING OF PROTEINS: ASSIGNMENT OF SECONDARY STRUCTURES BY ANTI-PEPTIDE ANTIBODIES

      Beyreuther, Konrad; Schulze-Gahmen, Ursula; Bieseler, Barbara; Prinz, Heinrich; Institute for Genetics, University of Cologne Weyertal 121, D-5000 Cologne 41, F.R. Germany (1987)
      We performed model studies towards assignment of &-turn and a-helices to protein primary structures with antibodies. Probing of a &-turn was attempted with anti-peptide antibodies directed against the &-turn (DPGQ) of a synthetic &-turn model-peptide (IVIVIDPGQTVTY) adopting the intended conformation &-sheet-&-turn-&-sheet. The specific anti-&-turn model-peptide antibodies have a three orders of magnitude higher affinity for the &-turn containing epitope than the control Gly-peptide (GsDPGQG,, ) of random coil structure. The antibody affinity for the &-turn region (DPGQ) increases from the primary to the hyperimmune response. Although the chosen &-turn sequence is similar to parts of the animal's own proteins, self-tolerance did not raise difficulties in generating antibodies against the R-turn model-peptide. Individual putative &-turn sequences of proteins may be probed by including their sequence between the two &-sheet cartridges of the &-turn model-peptide. Helix assignment was probed with synthetic model peptides of extended conformation including only the superimposed residues of a putative helix (every fourth residue) linked by a spacer amino acid residue (alanine throughout or the corresponding third residue of the sequence to be tested) in order to adjust the translation of the relevant residues of the model-peptide to the helical pitch. The anti-helix modelpeptide antibodies were shown by Western blotting to react in a sequence-specific manner with the corresponding model protein lactose permease of E.coli. "Conformational sequencing" i.e. sequence assignments of secondary structures by anti-peptide antibodies now seems feasable for &-turn regions and helices of proteins of known sequence.
    • PROTEIN ENGINEERING BY SITE DIRECTED MUTAGENESIS

      Winter, Greg; Carter, Paul; Bedouelle, Hugues; Wilkinson, Anthony J.; Fersht, Alan R.; MRC Laboratory of Molecular Biology, Hills Rd, Cambridge CBl 20H, England, and Department of Chemistry, Imperial College, London SW7 2AY, England (1987)
      The construction of mutations in the active site of the tyrosyl tRNA synthetase from Bacillus stearothermophilus has allowed us to deduce the relative impörtance of the substrate contacts to transition state binding. The feature dominating the energetics is the exchange reaction with water molecules: thus by deleting a poor H-bonding contact to the substrate we could increase the affinity of the enzyme for substrate. Furthermore by straining the polypeptide backbone by introducing a proline residue, we could improve the interaction of a histidine residue with the substrate. Thus enzymes affinities can be bettered by protein engineering in vitro.
    • SYNTHESIS OF 5-AZACYTOSINE 2’-DEOXYRIBONUCLEOSIDES, PROPERTIES AND BIOLOGICAL ACTIVITY

      Piskala, A.; Skutchan, J.; Cihak, A.; Vesely, J.; Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6, Czechoslovakia (1987)
      The effect of the o-D anomer Ia on L 1210 mouse leukemia cells was studied. This nucleoside inhibited the cell growth in vitro (IC55 about Lx 10-°m), and was also active in vivo in increasing the life span of mice with L1210 leukemia (800 to 1000 mg/kg) by 100%. It is Supposed that the action of the o-D anomer Ia is due to its conversion to Ib shown by HPCL performed on water solutions of both anomers. Similarly, the conversion of Ib to Ia was observed. Hydrolysis of 2'-deoxy-5-azacytidine (Ib) or its «-D anomer Ia with aqueous ammonia affords N-amidino-N’-(2-deoxyß- D-erythropentofuranosyl)urea formate (IVb). Cyclocondensation Of this compound with dimethylformamide dimethyl acetal leads to a mixture of anomeric nucleosides Ia and Ib. Stannic chloride catalysed condensation of blocked 2-deoxy-D-ribopyranose with silylated 5-azacytosine and subsequent methanolysis of the protected intermediates affords anomeric deoxyribopyranosyl nucleoside Va and vb which do not exhibit biological extivity. A series of 6-substituted deoxy nucleosides was prepared by direct glycosylation and the isocyanate procedure. 6-Amino-2’-deoxy-5-azacytidine prevents the growth of E. coli; the inhibition is completely reversed by natural purine bases and/or nucleosides.
    • SYNTHESIS AND CHARACTERIZATION OF A SET OF FOUR DODECADEOXYRIBONUCLEOSIDE UNDECAPHOSPHATES CONTAINING 0°-METHYLGUANINE OPPOSITE ADENINE, CYTOSINE, GUANINE, AND THYMINE

      Gaffney, B. L.; Marky, L. A.; Jones, R. A.; Department of Chemistry Rutgers, The State University of New Jersey New Brunswick, NJ 08903 USA (1987)
      A set of four self-complementary dodecanucleoside undecaphosphates, d[CGNGAATTC(O6Me)GCG] (1), where N = A, C, G, or T, has been synthesized by a phosphoramidite procedure. Each sequence forms a stable duplex, with a Im between 19 and 26°C lower than the Tm Of the "parent" molecule d(CGCGAATTCGCG). The lowest melting sequence is the N=T molecule; the overall order is N = C>A>G> T. Enus 0°-met hylation of guanine creates a region of localized instability in DNA regardless of the base opposite the lesion.
    • SYNTHETIC PEPTIDE VACCINES AND PROBLEMS ENCOUNTERED

      Birr, Christian; ORPEGEN , Med.-Molekularbiol. Forschungsgesellschaft m.b.H. Werderstrasse 35, 6900 Heidelberg F.R. Germany (1987)
      The development of suitable synthetic peptide vaccines depends both on the search for antigenic determinants in a given bacterial, viral or parasitic antigen and for immunologically acceptable carrier molecules for those antigenic determinants. This encounters a series of problems which are briefly discussed: synthesis of peptide antigens mainly made up from trifunctional amino acids, finding of antigenic determinants, which on immunization cause antibodies that recognize and precipitate not only the synthetic determinant but also the whole native antigen of which the determinants are just a few epitopic entities, construction of immunologically useful polymeric carrier molecules to which the synthetic antigenic determinants can be bonded in a well-defined and reproducible fashion, conformational considerations, which might become essential when a suitable epitope of a native antigen is substracted from its shape - inducing parent environment and, last not but not least, human health care aspects with respect to the applicability of results obtained from synthetic peptide vaccine developments for veterinarian diseases.
    • APPLICATIONS OF SYNTHETIC PEPTIDES

      Gutte, B.; Moser, R.; Klauser, S.; Leist, T.; Langen, H.; Epprecht, T.; Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland (1987)
      The revolutionary development of molecular biology during the past years has led to a strong interest in synthetic peptides. In this talk several important applications of synthetic peptides are discus sed. Peptides can be synthesized chemically in solution (1) or on solid supports (2,3). Up to a length of approximately 50 residues synthetic peptides have been obtained in (nearly) homogeneous form (4-6).
    • REGIO- AND STEREOSPECIFIC SYNTHESIS OF 7-DEAZAPURINE 2'-DEOXYRIBONUCLEOSIDES AND INCORPORATION OF NUCLEOSIDE ISOSTERES INTO OLIGONUCLEOTIDES

      Seela, F.; Driller, H.; Kehne, A.; Menkhoff, S.; Ott, J.; Winkeler, H.-D.; Universität Osnabrück, Laboratorium für Organische und Bioorganische Chemie, Fachbereich Biologie/Chemie, Barbarastr. 7, D-4500 Osnabrück (1987)
      2'-Deoxytubercidin and 7-deaza-2'-deoxyguanosine, isosteres of the parent nucleosides 2'-deoxyadenosine and 2'-deoxyguanosine have been synthesized via phase-transfer glycosylation of appropriately protected pyrrolo[2,3-d]pyrimidines with 1-chloro-2-deoxy-3 ,5-di-O-p-toluoy1-Derythro- pentofuranose via a regio- and stereospecific route. The nuclecside isosteres were provided with suitable protecting groups and converted into their O-3'-phosphoramidites. Application of these compounds in solid-support oligonucleotide synthesis yielded self-complementary oligomers with alternating d(TuT) or d(c’Gc) sequences. Additionally the incorporation of the 7-deazapurine 2'-deoxyribofuranosides into the Eco RI sequence was accomplished. Applying phosphite triester condensation in solution, (2',5')- and (3',5')-tubercidylyl-tubercidins were synthesized.