ABSTRACT
In order to increase the efficiency of use of automated DNA synthesizers (i.e. the number of oligomers prepared per day), we have devised and prepared novel phosphoramidite reagents that contain a linking group which, while stable under the normal synthesis conditions, is cleaved under basic conditions. When one of these linkers is introduced at the desired position in the synthesis of an oligonucleotide, subsequent detritylation enables the synthesis of a second oligonucleotides sequence upon the first. During deprotection of the oligonucleotide with ammonium hydroxide, the chain is cleaved at either side of the points of introduction of the novel reagent, generating two oligonucleotides free in solution. These reagents are of particular use in applications where oligomers are used in pairs (such as PCR, chemical synthesis of genes etc.) and means that an automated synthesis facility can be used more efficiently, without the need for operator intervention, after the working day is over.
Subject(s)
Amides , Indicators and Reagents , Oligonucleotides/chemical synthesis , Phosphoric Acids , Amides/chemistry , Ammonium Hydroxide , Autoanalysis , Base Sequence , Chromatography, High Pressure Liquid , Cloning, Molecular , Electrophoresis, Agar Gel , Humans , Hydrogen Bonding , Hydroxides , Methanol , Methylamines , Molecular Sequence Data , Molecular Structure , Phosphoric Acids/chemistry , Polymerase Chain Reaction , alpha 1-Antitrypsin/geneticsABSTRACT
The preparation of a reagent capable of reversibly attaching polyethylene glycol to proteins and the use of this material in modifying the plasminogen activators urokinase- and tissue-type plasminogen activator are described. The characterisation and the reversible nature of these protein-polymer conjugates are discussed, and some of the in vitro properties of these modified enzymes are explored.
Subject(s)
Polymers , Tissue Plasminogen Activator , Urokinase-Type Plasminogen Activator , Animals , Guinea Pigs , Maleic Anhydrides , Polyethylene Glycols , Tissue Plasminogen Activator/metabolism , Tissue Plasminogen Activator/pharmacokinetics , Urokinase-Type Plasminogen Activator/metabolism , Urokinase-Type Plasminogen Activator/pharmacokineticsABSTRACT
Treatment with conjugates of polysarcosine and grass pollen allergen extracts efficiently suppressed the induction of IgE responses in mice. The suppressive activity was shown to be allergen-specific and required covalent linking of the polysarcosine. Inhibitory effects could be overcome by booster injections of native allergen when these were given 3-4 weeks after treatment with conjugates. Administration of conjugates had only marginal effects on established IgE responses. The variance of these results with those of other studies on IgE suppression and the suitability of murine models for investigating reaginic antibody suppression are discussed.
Subject(s)
Adjuvants, Immunologic/pharmacology , Allergens/immunology , Immunoglobulin E/immunology , Peptides/immunology , Sarcosine/analogs & derivatives , Animals , Antibody Specificity , Immune Tolerance , Immunization, Secondary , Mice , Mice, Inbred Strains/immunology , Pollen/immunology , Sarcosine/immunologyABSTRACT
The ability of plasminogen to cause precipitation of soluble fibrin oligomers has been observed and certain features of the phenomenon investigated. The process is mediated by the lysine-binding sites and it appears that at least two such sites are required. Studies using radiolabelled plasminogen revealed that the precipitated material contained fibrin and plasminogen in a 2:1 molar ratio. Further plasminogen molecules are able to bind to the aggregate. The clotting of fibrinogen in the presence of plasminogen was studied using nephelometry. An enhancement by plasminogen of both the rate of clotting and the opacity of the clot was demonstrated. It is proposed that these effects are explicable in terms of a plasminogen-bridging model, in which the zymogen binds divalently between two monomer units of forming polymeric fibrin.
