Biomedical subjects
Publications and source records attributed to H Prydz.
Contrary to some recent reports DNA synthesis in isolated HeLa cell nuclei was not stimulated by the addition of low amounts of histones neither in the presence nor in the absence of cytosol. The individual histone fractions H1, H2A, H2B and H3 also failed to stimulated DNA synthesis.
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The subcellular localization of acarboxyprothrombin and the vitamin K-dependent carboxylation system has been studied using livers from vitamin K-deficient rats and found to be mainly in the membranes of the rough microsomal fraction. Bovine acarboxyprothrombin or fragment 1 thereof were inactive as substrates in the rat carboxylating system.
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The effect of the inhibitor aphidicolin on DNA synthesis in isolated nuclei from HeLa cells and on the activities of partially purified DNA polymerases has been tested. Aphidicolin inhibited DNA synthesis and DNA polymerase alpha very efficiently whereas DNA polymerases beta and gamma were insensitive to the drug. The results indicate that DNA polymerase alpha is the polymerase active during elongation as well as in the gapfilling process of discontinuous DNA synthesis.
An immunoradiometric assay for tissue thromboplastin has been established. Blood levels in 6 patients undergoing total hip replacement have been determined. In 3 patients, high levels of circulating apoprotein III were found at various stages of the operation, showing a release of tissue thromboplastin chiefly during impaction of the prosthesis into the femoral bone. The other 3 patients had low or undetectable levels.
Injection of bacterial lipopolysaccharide into pregnant mice resulted in fibrinogen accumulation, thrombosis and haemorrhage in the placental tissue and foetal death. Depletion of circulating fibrinogen by a thrombin-like enzyme from the venom of Malayan pit viper, Arvin, prevents foetal death. Foetal protection was also obtained by treating the mothers with a preparation of phospholipase C from Bacillus cereus known to inactivate tissue thromboplastin. It is suggested the lipopolysaccharide causes foetal death by inducing thrombosis as a consequence of activation of placental thromboplastin.
Certain agents that induce spreading of or increased content or release of lysosomal enzymes from human monocytes also induce a dramatic increase in tissue thromboplastin content of the cells. The increase is inhibited by cycloheximide and actinomycin D and does not appear in granulocyte, lymphocyte or platelet populations treated in the same way. The effect of several other substances on the development of monocyte tissue thromboplastin activity is reported.
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A simple three-step method was established for the purification of NAD(P)H dehydrogenase (quinone) ('DT-diaphorase', EC 1.6.99.2) from rat liver by affinity chromatography with a recovery of above 50%. The final enzyme preparation was purified about 750-fold and was electrophoretically homogeneous. Gel filtration showed that the enzyme had a mol.wt. of about 55 000, and one molecule of FAD was found per 55 000 mol.wt. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave a mol.wt. of about 27 000. Two N-terminal amino acids, asparagine/aspartic acid and glutamine/glutamic acid, were found in about equal yield, suggesting the presence of two non-identical polypeptide chains in the enzyme. NAD(P)H dehydrogenase was selectively removed by this affinity-chromatographic method from a microsomal carboxylation system. The system, which was solubilized by detergent and is dependent on vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone or analogues with other side chains), lost its activity on the removal of the enzyme. The activity can be completely restored to the system by adding purified cytoplasmic NAD(P)H dehydrogenase or by using the quinol form of vitamin K1 (2-methyl-3-phytyl-1,4-naphthaquinol).
The macrophage is the characteristic cell type in chronic inflammatory reactions, in the rheumatoid synovium, as in other sites. When macrophages are activated, considerable synthesis of enzymes and other proteins occurs. Macrophages can be activated by (i) products of activated lymphocytes, (ii) immune complexes and (iii) the complement cleavage product C3b. Among the many consequences of macrophage activation are (i) secretion of hydrolytic enzymes, (ii) cleavage of C3 into C3a, which is cytolytic, and C3b, (iii) production of tissue thromboplastin, a powerful procoagulant, and (iv) formation of polyamine oxidase, which in the presence of appropriate substrates generates factors that lyse or limit the proliferation of tumour cells, lymphocytes and micro-organisms. The relevance of these observations to the pathogenesis of chronic inflammatory reactions is discussed.
A solid-phase immunoradiometric assay for tissue thromboplastin (factor III) has been established based on its displacing effect on the binding of 125I-labelled factor III-antibodies to polyvinyl tubes coated with the purified protein component of factor III (apoprotein III). By this method circulating tissue thromboplastin can be detected in experimental animals receiving infusions of crude or purified tissue thromboplastin and in patients undergoing major orthopaedic surgery.
The effect of nalidixic acid, rifampicin and chloramphenicol on the synthesis of phospholipase C (EC 3.1.4.3) has been studied in washed Bacillus cereus cells resuspended in nutrient broth. In the absence of inhibitors, the synthesis showed a biphasic pattern. No synthesis of release of enzyme was found in the presence of chloramphenicol. When rifampicin was added, phospholipase C synthesis continued for 10-15 min. Nalidixic acid, at concentrations which inhibited DNA synthesis completely, permitted the synthesis of phospholipase C at the same rate and for a similar length of time as rifampicin.
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Replicative intermediates have been studied in intact HeLa cells and in nuclei isolated from such cells. In whole cells the smallest DNA (primary DNA pieces) observed after pulse labelling with [3H]thymidine were 90-160 nucleotides long, and the size of the molecules in this class of DNA did not increase with increasing pulse length. Some increase in size was, however, observed when cells were pulse labelled at 25 degrees C instead of 37 degrees C. Chase experiments using nuclei from pulse-labelled cells suggested that the primary DNA pieces could be chased rapidly into DNA of high molecular weight (30-70 S, corresponding to a molecular weight of 0.7 - 10(7)-6.4-10(7)). Longer chases showed that the label eventually accumulated in DNA with s values greater than 150 S. In isolated nuclei the primary DNA pieces after a 1 min pulse at 37 degrees C were approximately 200 nucleotides long. Primary pieces of this size were also rapidly chased into the 30-70 S region. However, during longer pulses in vitro a fraction of the primary DNA pieces grew beyond their normal size to reach a size of up to 2000-3000 nucleotides before being attached to the 30-70 S molecules.
Some immunological studies on prothrombin fragment 1 from bovine prothrombin and its warfarin-induced precursor acarboxyprothrombin are reported. Based on the results, a rapid and simple immunoadsorption method for the isolation of prothrombin fragment 1 in good yield has been established. The method exploits the conformational change induced in the fragment by removal of Ca2+. The principle may be applicable to other gamma-carboxyglutamyl-containing proteins or fragments therof.