Confirmatory evidence for the existence of a multienzyme complex of DNA precursor pathways in mammalian cells was obtained. Using neutral sucrose gradient centrifugation of cell lysates we found that at least five enzymes involved in DNA precursor metabolism in uninfected. S-phase BHK-cell fibroblasts cosediment at a common rate, indicative of a multienzyme complex. The enzymes include DNA polymerase thymidine kinase, ribonucleotide reductase, dihydrofolate reductase, and NDP-kinase. This complex was partially, but not completely, disrupted when lysates from GO-phase cells were centrifuged. Using lysates from cells infected with herpes simplex virus (HSV) type I some of the virus-induced ribonucleotide reductase and a minor proportion of the HSV-thymidine kinase cosedimented rapidly. The virus-induced DNA polymerase sedimented independently near the middle of the gradient, in contrast to the behaviour of the host polymerase. The enzyme associations observed were disrupted by NaCl or by inclusion of ethylenediamine tetraacetic acid during the cell lysis procedure, instead of the usual EGTA. These results indicate the importance of ionic forces in maintaining the enzyme complexes. The bulk of the DNA and the RNA present in the lysates did not sediment at the same rate as the complexes, showing that the enzymes were not simply adhering nonspecifically to these polyanions. Newly synthesised radiolabeled DNA (15 min pulse with [3H]thymidine) was not preferentially associated with the enzymes, but some functional DNA was evident in the enzyme complex fraction from the uninfected S-phase cells. DNA polymerase activity in this fraction did not require, nor was it stimulated by, exogenous "activated" DNA. Added DNA primer-template was required, however, for maximal activity of the polymerase in gradient fractions derived from GO-phase cells and from HSV-infected cells. No evidence for channeling of ribonucleotide precursors into DNA of permeabilized cells (uninfected or HSV-infected) was detected. Most rCDP was incorporated into RNA. In the uninfected, S-phase cells about 10 pmol/10(6) cells/90 min of rCDP residues was incorporated into DNA compared with 120 pmol/10(6) cells/90 min when radiolabeled dCTP was used. Nonradioactive dCTP present in equimolar concentration in the incubation with labeled rCDP did not, however, diminish the incorporation of label from the ribonucleotide. In permeabilized HSV-infected cells incorporation of radiolabel from rCDP into DNA was barely detectable.
A case of cutaneous cryptococcosis (encapsulated strain) in a 67-year-old female, with no evidence of immune suppression (normal cell surface marker analysis and mitogen proliferation studies) and which responded to treatment with oral fluconazole is reported. To date her clinical progress remains satisfactory after 12 months of follow-up.
A rapid method has been developed for measuring the binding capacity of asbestos and other mineral fibres for environmental carcinogens. Benzo(a)pyrene (B(a)P), nitrosonornicotine (NNN), and N-acetyl-2-aminofluorene (NAAF) were assayed in the presence of Canadian grade 4T30 chrysotile, chrysotile A, amosite, crocidolite, glass microfibres, glasswool, attapulgite, and titanium dioxide. Chtysotile binds significantly more carcinogens than the other mineral fibres. This binding assay is reproducible with coefficients of variation of less than 8% and 6% respectively for inter and intra assay. The influence of pH was also studied, and there is good correlation between the carcinogen binding and the charge of the tested mineral fibres. The in vitro cytotoxicity on macrophage like cell line P388D, and the haemolytic activity of various mineral fibres were also measured; a good correlation was found between the binding capacity and the cytotoxicity of tested mineral fibres on P388D, cells. These results give some explanations for the reported synergism between exposure to asbestos and the smoking habits of workers.The potentially harmful effects of all types of respir- Owing to its binding capacity for carcinogens present in the environment or in tobacco smoke, asbestos may be a cancer promoter. It is therefore necessary to identify which mineral fibres interact with carcinogens to induce neoplasia. The present data describe the binding capacity of mineral fibres for three carcinogens and the correlation with some in vitro biological assays.
620th MEETING. DUBLIN 265In order to prepare pure enzyme before gene cloning the proteins were separated by fast protein liquid chromatography and used to prepare monoclonal antibodies to papaya proteinase A and B.Crude spray-dried latex from Powell and Scholefield was dialysed against 1,3-diaminopropane and the enzyme isolated by the method of Goodenough & Owen (1986) using 6 mM-diaminopropane as buffer and a gradient of 4 mM/min of NaCI. The use of an anion-exchange column gave a good separation of the proteins, and fractions containing papaya proteinase A and B were freeze-dried and redissolved in phosphate-buffered saline (0.1 ~-NaC1/0.06 M-phosphate, pH 7.3).Balb/c mice were injected subcutaneously with 50pg of a mixture of the redissolved protein in 200p1 of Freunds complete adjuvant. Six weeks later, after an intravenous booster injection, fusion of the spleen cells and mouse myeloma cells, P3 x 63-Ag 8-653 was obtained. Eight hybridomas were shown to have immunological activity when reacted against mixed papaya proteinase A and B. Western immunoblotting and enzyme-linked immunosorbent assays were as described by Goodenough et al. (1986).Two lines, PAP 8 and PAP 7, were shown to be specific for papaya proteinases A and B respectively. The reaction of PAP 7 and PAP 8 is shown in Fig. 1 and it can be easily seen that there is a specific reaction with the papaya proteinases.When the papaya proteinases are separated by fast protein liquid chromatography the individual members can be prepared in a nearly pure form. The activity of the prepara-tions of proteinases A and B towards casein is the same qualitatively, although A is more active quantitatively. It is conceivable that activity of A is contaminating B and that B only has a lysozymal activity. However, we have determined the M , of B as 28 000 and the PI as > 1 1.1. These are widely different from the properties of most lysozymes. The fact that monoclonal antibodies that are specific for A and B can be prepared so easily indicates that at least some of the quaternary structure of the molecule is quite different in structure.We have used the monoclonal antibodies to purify quantities of A and B for sequencing and crystallographic analysis. It is hoped to be able to also isolate the DNA coding for this interesting family of proteinases.
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