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2.
J Med Chem ; 39(23): 4608-21, 1996 Nov 08.
Article in English | MEDLINE | ID: mdl-8917650

ABSTRACT

The active metabolite (2) of the novel immunosuppressive agent leflunomide (1) has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. A series of analogues of the active metabolite 2 have been synthesized. Their in vivo biological activity determined in rat and mouse delayed type hypersensitivity has been found to correlate well with their in vitro DHODH potency. The most promising compound (3) has shown activity in rat and mouse collagen (II)-induced arthritis models (ED50 = 2 and 31 mg/kg, respectively) and has shown a shorter half-life in man when compared with leflunomide. Clinical studies in rheumatoid arthritis are in progress.


Subject(s)
Acrylamides/chemical synthesis , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Oxidoreductases Acting on CH-CH Group Donors , Oxidoreductases/antagonists & inhibitors , Acrylamides/pharmacokinetics , Acrylamides/therapeutic use , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Arthritis, Experimental/drug therapy , Dihydroorotate Dehydrogenase , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/therapeutic use , Female , Hypersensitivity, Delayed , Kinetics , Magnetic Resonance Spectroscopy , Male , Mice , Mice, Inbred DBA , Rats , Rats, Wistar , Spectrophotometry, Infrared , Structure-Activity Relationship
3.
J Biol Chem ; 270(38): 22467-72, 1995 Sep 22.
Article in English | MEDLINE | ID: mdl-7673235

ABSTRACT

A protein with high affinity (Kd 12 nM) for the immunomodulatory compound A77 1726 has been isolated from mouse spleen and identified as the mitochondrial enzyme dihydroorotate dehydrogenase (EC 1.3.3.1). The purified protein had a pI 9.6-9.8 and a subunit Mr of 43,000. Peptides derived from the mouse protein displayed high microsequence similarity to human and rat dihydroorotate dehydrogenase with, respectively, 35 and 39 out of 43 identified amino acids identical. Dihydroorotate dehydrogenase catalyzes the fourth step in de novo pyrimidine biosynthesis. The in vitro antiproliferative effects of A77 1726 are mediated by enzyme inhibition and can be overcome by addition of exogenous uridine. The rank order of potency of A77 1726 and its analogues in binding or enzyme inhibition was similar to that for inhibition of the mouse delayed type hypersensitivity response. It is proposed that inhibition of dihydroorotate dehydrogenase is an in vivo mechanism of action of the A77 1726 class of compounds. This was confirmed using uridine to counteract inhibition of the murine acute graft versus host response.


Subject(s)
Aniline Compounds/metabolism , Hydroxybutyrates/metabolism , Oxidoreductases Acting on CH-CH Group Donors , Oxidoreductases/metabolism , Amino Acid Sequence , Aniline Compounds/pharmacology , Animals , Binding Sites , Cell Division/drug effects , Crotonates , Dihydroorotate Dehydrogenase , Growth Inhibitors/chemistry , Hydroxybutyrates/pharmacology , Mice , Microsomes/metabolism , Mitochondria/metabolism , Molecular Sequence Data , Molecular Structure , Nitriles , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/chemistry , Spleen/metabolism , Toluidines , Uridine/pharmacology
7.
Eur J Biochem ; 217(3): 965-71, 1993 Nov 01.
Article in English | MEDLINE | ID: mdl-8223654

ABSTRACT

Recent data suggest that botulinum type-B neurotoxin is a protease which acts on vesicle-associated membrane protein, isoform 2 (VAMP-2). In this report, botulinum type-B neurotoxin is shown to cleave a synthetic fragment (HV62) of VAMP-2, corresponding to the bulk of the hydrophilic domain (amino acids 33-94). The neurotoxin acts at a single site between Gln76 and Phe77. Little or no proteolytic activity by botulinum type-B neurotoxin was observed with peptides containing 7, 10 or 20 amino acids spanning the site of cleavage. The proteolytic action of neurotoxin was strongly inhibited by EDTA and o-phenanthroline whereas captopril and phosphoramidon were ineffective. A series of model peptide substrates were synthesised in order to define the smallest VAMP-2 fragment to be cleaved by botulinum type-B neurotoxin. Data obtained from these substrates suggest that the neurotoxin belongs to a novel class of zinc-endoprotease; more than 12 amino acid residues are required on both the NH2- and COOH-terminal side of the cleavage site for optimal proteolytic activity. The results demonstrate that no other components of cellular vesicles are required for the specific action of the neurotoxin on VAMP-2. The data further show that the highly specific action of the neurotoxin is not dictated solely by the properties of the amino acid residues at the cleavage site but is also dependent on amino acid sequences distal to its site of action.


Subject(s)
Botulinum Toxins/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Peptide Fragments/metabolism , Amino Acid Sequence , Humans , Hydrolysis , Membrane Proteins/chemistry , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , R-SNARE Proteins
9.
JAMA ; 269(2): 216, 1993 Jan 13.
Article in English | MEDLINE | ID: mdl-8417238
10.
Biotechniques ; 13(3): 450-3, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1389178

ABSTRACT

The prevention and control of biohazards arising from the use of laboratory instruments have become increasingly important in clinical and research applications. Centrifuges can be susceptible to contamination because of intense wear on primary containers (specimen tubes and bottles), worn O-ring container seals, or rotors and buckets lacking tight seals. A recent study by the Center for Applied Microbiology and Research, Porton Down, UK, has determined the biological safety of certain rotors in various speed ranges. This paper presents and discusses these findings.


Subject(s)
Accident Prevention , Accidents, Occupational/prevention & control , Centrifugation , Containment of Biohazards , Equipment Contamination , Laboratory Infection/prevention & control , Research Personnel , Aerosols , Air Microbiology , Bacillus subtilis/isolation & purification , Centrifugation/instrumentation , DNA, Recombinant , Escherichia coli/isolation & purification , Humans
13.
Bioseparation ; 3(5): 267-83, 1992.
Article in English | MEDLINE | ID: mdl-1369426

ABSTRACT

Bacterial protein toxins and their fragments have been isolated and purified for various reasons, including the development of efficient vaccines and for methods of identification of bacterial agents causing disease. This activity continues today but a new area of bacterial protein toxin research has recently emerged. Since it was shown that toxin molecules comprise several types of biological activity within their structural domains, it was suggested to use these domains (and their combinations) as biochemical tools for developing novel agents for disease imaging and and/or relieving. In this way eukaryotic cell-receptor specific fusion toxins have been developed to prevent malignancy in human. While human clinical trials of these preparations have only recently begun, the preliminary clinical findings are promising. Also fusion proteins which combine independent immunodominant epitopes from different antigens have also been developed thus opening a way for the generation of new vaccines for both human and veterinary use. Receptor binding fragments of microbial toxins when combined with other molecules may be useful in delivering these molecules into the cell. In this way novel agents may be developed with a potential for inducing specific changes at the molecular level for the correction of metabolic disorders causing human and animal diseases. Bacterial protein toxins such as anthrax, botulinum, cholera, pertussis and tetanus for which considerable progress has been achieved in structure-function analysis are promising candidates for such research. Particularly exciting appears the idea of extending this research to the cells of the nervous system, exploiting the unique specificity of the botulinum or tetanus toxin fragments which may bring long desired methods for treatment of various disorders of the nervous system. Data on functional domains of these toxins as well as methods of purification of the whole toxins and their fragments are considered in this review as they form a base for their further structure-function analysis and engineering applications.


Subject(s)
Antigens, Bacterial , Bacterial Toxins/isolation & purification , Exotoxins/isolation & purification , Animals , Bacillus anthracis/metabolism , Botulinum Toxins/isolation & purification , Cholera Toxin/isolation & purification , Chromatography, Liquid , Humans , Tetanus Toxin/isolation & purification , Virulence Factors, Bordetella/isolation & purification
14.
J Neurol ; 239(1): 16-20, 1992 Jan.
Article in English | MEDLINE | ID: mdl-1311751

ABSTRACT

The neurotoxins produced by Clostridium botulinum are the most potent acute toxins known and are the causative agents of the neuroparalytic disease botulism. The toxins act primarily at peripheral cholinergic synapses by blocking the evoked release of the neurotransmitter acetylcholine. There are seven distinct serotypes of toxin. All are polypeptides of Mr about 150 kDa that have similar structure and pharmacological action. In their most active forms the toxins exist as dichain molecules in which a heavy (H) chain is linked by disulphide bonding to a light (L) chain. The H chain is believed to be associated with the highly specific and avid binding of toxin to the motor nerve end plates and also with the process of internalisation of the toxin. The toxic activity appears to be associated with the L chain which blockades the calcium-mediated release of acetylcholine, probably by interfering at the molecular level with the mechanisms whereby neurotransmitter-containing vesicles merge with the plasmalemma. The type A toxin is now used therapeutically to treat a variety of conditions involving involuntary muscle spasm. The therapeutic toxin is a neurotoxin-haemagglutinin complex isolated from cultures of C. botulinum. A controlled manufacturing process has been developed for the therapeutic toxin which is specially formulated to give a freeze-dried product having good stability.


Subject(s)
Botulinum Toxins , Clostridium botulinum/metabolism , Acetylcholine/metabolism , Animals , Botulinum Toxins/chemistry , Botulinum Toxins/classification , Botulinum Toxins/isolation & purification , Botulinum Toxins/pharmacology , Botulinum Toxins/therapeutic use , Botulism/microbiology , Botulism/physiopathology , Clostridium botulinum/pathogenicity , Depression, Chemical , Endocytosis , Food Contamination , Humans , Molecular Weight , Neuromuscular Junction/drug effects , Neurons/drug effects , Neurons/metabolism , Receptors, Cholinergic/metabolism , Spasm/drug therapy
15.
J Chem Technol Biotechnol ; 50(2): 167-80, 1991.
Article in English | MEDLINE | ID: mdl-1367227

ABSTRACT

A 225 dm3 pilot-plant bioreactor system has been designed and constructed that is suitable for biohazardous fermentations. The design enables operation at containment levels above the requirements of good industrial large-scale practice (GILSP) without secondary containment of the whole plant. The main biosafety features of the systems include the use of steam barriers on O-ring seals, supply lines and stirrer seals, multiple O-ring seals, piping of condensate lines and pressure relief systems to a 'kill tank', double filtration of inlet and off gases and a mobile isolation unit that allows localised containment of sample valve and probe entry ports. The fermenter can, with minor modifications, be operated as a bottom-or top-stirred reactor for the culture of microbial or animal cells, or as an airlift reactor. The design offers considerable flexibility that could prove cost-effective for process development and production. The relevance of the various design features to enable bioreactor operations at pilot-plant scale to be carried out in compliance with current guidelines for large-scale culture of recombinant microorganisms and microbial pathogens is discussed.


Subject(s)
Biotechnology/instrumentation , Fermentation , Animals , Biotechnology/methods , Biotechnology/standards
16.
Biochem J ; 268(1): 123-8, 1990 May 15.
Article in English | MEDLINE | ID: mdl-2188647

ABSTRACT

1. A large-scale purification procedure has been developed for Clostridium botulinum type F neurotoxin. Commencing with 160 litres of bacterial culture, 101 mg of purified type F neurotoxin with a specific toxicity of 2 x 10(7) mouse LD50 (median lethal dose).mg-1 were obtained. 2. Purified type F neurotoxin was labelled to high specific radioactivity (900-1360 Ci/mmol) without loss of biological activity using a chloramine-T procedure. Of the two neurotoxin subunits, the heavy chain was preferentially radiolabelled. 3. Radiolabelled type F neurotoxin displayed specific saturable binding to rat synaptosomes. At least two pools of acceptors were evident: a low content of high-affinity acceptors sites [KD approximately 0.15 nM; Bmax (maximal binding) 20 fmol/mg] and a larger pool of lower-affinity sites (KD greater than 20 nM; Bmax greater than 700 fmol/mg). Both pools of acceptors were sensitive to trypsin and neuraminidase treatment, which suggests that protein and sialic acid residues are components of the synaptosomal acceptors. 4. Experiments investigating competition among botulinum neurotoxin types A, B, E and F for acceptors on rat brain synaptosomes showed that type F neurotoxin binds to acceptor molecules which are completely distinct from those of the other three neurotoxins.


Subject(s)
Botulinum Toxins/isolation & purification , Cerebral Cortex/metabolism , Clostridium botulinum/metabolism , Synaptosomes/metabolism , Animals , Botulinum Toxins/metabolism , Botulinum Toxins/pharmacology , Chromatography , Electrophoresis, Polyacrylamide Gel , Iodine Radioisotopes , Rats , Synaptic Membranes/metabolism
17.
Appl Environ Microbiol ; 56(4): 1067-72, 1990 Apr.
Article in English | MEDLINE | ID: mdl-2339869

ABSTRACT

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.


Subject(s)
Chromatography, Affinity/methods , Enterotoxins/isolation & purification , Coloring Agents , Isoelectric Point , Staphylococcus aureus/analysis , Triazines
18.
Agents Actions ; 29(3-4): 328-32, 1990 Mar.
Article in English | MEDLINE | ID: mdl-2339672

ABSTRACT

Rats were sensitized by i.d. injection in the base of the tail with Freund's complete adjuvant (FCA) and were challenged i.d. in the dorsal skin with mycobacterial antigen. The 24 hour dermal delayed-type hypersensitivity (DTH) response increased up to 10 days after FCA injection followed by a decrease by day 15 which coincided with the development of adjuvant arthritis (AA). Drug studies were performed, using a 4-day dosing schedule, on optimal DTH elicited on day 10, suboptimal DTH elicited on day 15, and AA (day 16). Cyclosporine, leflunomide and prednisolone significantly inhibited day 10 DTH and AA with no effect on day 15 DTH. Indomethacin and tiaprofenic acid significantly inhibited AA with no effect on either DTH response. Chloroquine, levamisole, D-penicillamine, diazepam and RU38468 had no significant effect on DTH or AA. These findings suggest a complex temporal relationship between AA, DTH and drug actions.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Arthritis, Experimental/immunology , Arthritis/immunology , Hypersensitivity, Delayed , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antigens, Bacterial/immunology , Cyclosporins/pharmacology , Freund's Adjuvant , Indomethacin/pharmacology , Isoxazoles/pharmacology , Kinetics , Leflunomide , Male , Mycobacterium/immunology , Propionates/pharmacology , Rats , Steroids
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