Biomonitoring of arylamines: haemoglobin adducts of aniline derivatives

Aromatic amines are important intermediates in industrial manufacturing. They are used in a large number of products, such as pesticides, dyes, plastics and pharmaceuticals. The parent arylamines can be metabolically released from these arylamine-based compounds and form DNA and protein adducts after N-oxidation to N-hydroxy arylamines. Aromatic amine derivatives, including the industrial intermediates acetoacetanilide, acetoacet-m-xylidide and N-ethylaniline, were examined for their ability to form Hb adducts in rats as potential biomarkers of exposure. The haemoglobin binding indices (HBI=binding [mmol mol^-1 Hb]/dose [mmol kg^-1 body weight]) of the arylamines were determined 24 h after oral administration to female Wistar rats. The precipitated haemoglobin was dissolved in 0.1 M sodium hydroxide in the presence of internal standards. After hexane extraction the released arylamines were analysed by gas chromatography-mass spectrometry (GC-MS). For aniline released from acetoacetanilide an HBI of 15 and for 2,4-dimethylaniline released from acetoacet-m-xylidide an HBI of 0.129 were determined. The HBIof aniline released from N-ethylaniline was 45.     

Functional role of lipid metabolism in activated T-lymphocytes

Exogenous long-chain fatty acids are readily taken up by unstimulated lymphocytes derived from the thymus of calves or rabbits and esterified to complex lipids, primarily phospholipids and triacylglycerols. Compared to saturated fatty acids, unsaturated fatty acids are incorporated preferentially. Furthermore, unsaturated fatty acids are transferred from triacylglycerols to phospholipids. The transfer cannot be observed with palmitic acid. With regard to individual phospholipid species, oleic acid and linoleic acid are found primarily in phosphatidylcholine. Arachidonic acid, however, is transferred to phosphatidylethanolamine and phosphatidylinositol as well. This suggests an arachidonic-specific transfer between individual phospholipids. Stimulation of the cells with the mitogen concanavalin A results in an enhanced incorporation of the fatty acids and an enhanced transfer from triacylglycerols to phospholipids. Triacylglycerols may thus be regarded as a labile intracellular storage pool that may be activated upon mitogenic stimulation.     

Pitfalls in measuring fluorescence polarization in mitogen-stimulated T-lymphocytes

Fluorescence polarization measurements with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed to detect changes in the fluidity of plasma membranes from T-lymphocytes stimulated with mitogens. When the cells were incubated with succinyl-concanavalin A an increase in fluorescence polarization was observed. This, however, could be shown to be due to the interaction of the mitogen with the label DPH and did not reflect changes in the plasma membrane. In purified plasma membranes a decrease rather than an increase of fluorescence polarization was observed.     

Regulation of prostaglandin synthesis by protein kinase C in mouse peritoneal macrophages.

Resident mouse peritoneal macrophages synthesized and released prostaglandins (PGs) when challenged with 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol (DiC8). Both stimuli were found to activate Ca2+/phospholipid-dependent protein kinase C (PKC). 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine ('H-7') and D-sphingosine, known to inhibit PKC by different mechanisms, were able to decrease the PKC activity of macrophages in a dose-dependent manner. Addition of either PKC inhibitor decreased PG synthesis and also the release of arachidonic acid (AA) from phospholipids induced by TPA or DiC8. Simultaneously TPA or DiC8 also decreased incorporation of free AA into membrane phospholipids of macrophages. AA incorporation could be restored, however, by pretreatment with the PKC inhibitors. Our results demonstrate an involvement of PKC in the regulation of PG synthesis in mouse peritoneal macrophages and provide further evidence that reacylation of released fatty acids may be an important regulatory step.     

Characterization of the interleukin-1-induced tyrosine phosphorylation of a 41-kDa plasma membrane protein of the human tumor cell line K 562

A 41-kDa protein, which was specifically phosphorylated upon incubation with natural purified murine interleukin 1, was recently identified by us [Martin, M., Lovett, D. H. and Resch, K. (1986) Immunobiology 171, 165-169] in highly purified plasma membranes from the human tumor cell line K 562. An in vitro assay was used to investigate and characterize the phosphorylation induced by interleukin 1, possibly involved in signal transduction and generation. Plasma membranes were incubated with radiolabeled ATP in the presence of purified natural murine interleukin 1, or recombinant human interleukin 1 alpha and the pattern of phosphoproteins was studied after separation by SDS/PAGE and subsequent autoradiography. A 41-kDa protein (pp41) was specifically phosphorylated on a tyrosine residue in the presence of interleukin 1 in a dose- and time-dependent manner. The protein showed a weak background phosphorylation in the absence of monokine. Phosphorylation took place very efficiently at 0 degrees C, whereas phosphatases were not active at that temperature. At 37 degrees C, a rapid dephosphorylation was observed which was inhibited specifically by Zn2+ and vanadate. The interleukin-1-specific induction of the phosphorylation could also be observed after detergent solubilization of the plasma membranes. Affinity labeling with an ATP analogue revealed an ATP-binding and cleaving site at 41 kDa. Interleukin 1 did not induce the phosphorylation of p41 in plasma membranes obtained from a subclone of K 562, which did not respond to interleukin 1 with growth inhibition, as was reported recently for the K 562 mother line [Lovett, D. H., Kozan, B., Hadam, M., Resch, K. and Gemsa, D. (1986) J. Immunol. 136, 340-347]. These data suggest that the interleukin-1 receptor is functionally linked to a protein-tyrosine kinase, which is implicated in its biological function.     

The membrane dialysis bioreactor with integrated radial-flow fixed bed —a new approach for continuous cultivation of animal cells

A hybridoma cell was cultivated continuously in a membrane dialysis bioreactor with an integrated radial-flow fixed bed consisting of porous Siran^® carriers over a period of 6 weeks. Antibodies accumulated to an average of 100 mg l^?1, approx. 10 times more than in fixed bed cultures without dialysis membrane. Serum costs could be reduced about 85% due to an appropriate feeding strategy. Siran^® carriers with 3–5 mm diameter showed an advantage compared to those with 1–2 mm diameter. For the 3–5 mm carrier the specific glucose uptake rate and the MAb production rate were constant, if the velocity was between 0.09 mm s^?1 and 0.75 mm s^?1. At higher velocities cells are washed out of the bed. Furthermore antibody consistency and cell stability were verified in long-term cultivations over a period of 96 days. From an estimation of the antibody concentration reachable with the reactor concept under optimal conditions a concentration 45 times higher compared to axial-flow fixed bed reactors and 11 times higher compared to stirred tank reactors can be expected.     

Induction of prostanoid synthesis in human platelets by the late complement components C5b-9 and channel forming antibiotic nystatin: inhibition of the reacylation of liberated arachidonic acid

Treatment of human platelets by the purified late complement components C5b-9 results in a dose- and time-dependent release of prostaglandin E (PGE) and thromboxane B2 (TXB2). To study the mechanism underlying the complement-induced prostanoid synthesis, we examined whether C5b-9 affected the enzyme acyl-coA:lysolecithin acyltransferase (E.C.2.3.1.2.3) that catalyzes the reinsertion of liberated arachidonic acid, the precursor molecule of the prostanoids. With C5b-9 doses sufficient to induce prostanoid synthesis, the activity of lysolecithin acyltransferase, measured as conversion of lysophosphatidyl choline to phosphatidyl choline, was inhibited. For comparison, another channel-forming substance, nystatin, was studied. Nystatin had an effect similar to C5b-9: PGE and TXB2 release was stimulated, whereas acyltransferase activity was inhibited. These finding support the concept that inhibition of lysolecithin acyltransferase might be the prerequisite for prostanoid production.