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.     

Isolation and characterization of a 40-kDa cyclophilin-related protein.

Major and minor isoforms of cyclophilin (CyP-18), a 17.8-kDa protein with peptidyl-prolyl cis-trans isomerase activity, comprise the primary intracellular binding proteins for cyclosporin A. Additional CyP-like proteins with approximate molecular masses of 22 (CyP-22) and 40 kDa (CyP-40) have been recovered from the soluble fraction of calf brain along with CyP-18 by adsorption onto a cyclosporin A affinity column and elution with cyclosporin A. Based on a limited number of peptide sequences from CyP-22, it appears that we have isolated from tissue CyPB, a protein whose sequence was deduced previously from cloned cDNA. The 40-kDa protein was separated from CyP-18 and CyP-22 on a molecular sieving column. Isoelectric focusing of CyP-40 yielded two bands at pI 5.3 and 5.5, in contrast to the basic pI values of CyP-18. Some tryptic peptides from CyP-40 were found to be highly homologous but not identical to bovine CyP-18; others were not significantly homologous to CyP-18 or any other protein in the data base. Unlike the major and minor isoforms of Cyp-18, monospecific polyclonal anti-CyP-18 antibodies did not cross-react with CyP-22 and CyP-40. Likewise, anti-CyP-40 serum minimally cross-reacts with CyP-18 and CyP-22. Cyp-40 possesses peptidyl-prolyl cis-trans isomerase activity which is less sensitive to inhibition by cyclosporin A (IC50 = 300 nM) than is CyP-18 (IC50 = 20 nM).     

Separation of hepatocytes of different acinar zones by flow cytometry

Hepatocytes in the proximal (zone 1) and distal (zone 3) regions of the liver acinus are selectively stained by perfusion of the isolated rat liver with 0.2-20 microM acridine orange (AO). After 10-60 min of anterograde perfusion, AO fluorescence is visible in zone 1 cells, whereas retrograde perfusion stains cells of zone 3. In this paper, we describe a technique to isolate a mixed population of fluorescent and nonfluorescent hepatocytes (cells from all acinar zones, which do not loose the zone specific AO labeling) and to separate these cells according to their zonal origin by fluorescence activated cell sorting. The zonal populations obtained were either fluorescent or nonfluorescent (purity greater than 95%). Separated cell fractions differed in their enzyme content (5' nucleotidase, succinate-dehydrogenase, beta-glucuronidase). An unidentified AO metabolite, which is not found in bile after retrograde perfusion (not formed in zone 3 cells), is also absent after retrograde perfusion in sorted fluorescent cells (zone 3 cells), indicating zonal purity of sorted cells.     

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.     

Elektronenmikroskopische Untersuchungen zur Lipid-Speicherung in den Nierentubuli von Drosophila melanogaster

Zusammenfassung In den Zellen der Nierentubuli (Malpighische Gefäße) von Drosophila melanogaster werden Lipide in der gleichen Weise wie das 3-Hydroxykynurenin gespeichert. Diese Substanzen werden in Erweiterungen des endoplasmatischen Retikulums akkumuliert. Bei älteren Larven verschwinden diese Lipidtropfen. Dabei legen sich entweder Mitochondrien um die Depots oder sie werden von Membranstapeln des endoplasmatischen Retikulums umgeben und abgebaut. Die funktionelle Bedeutung dieser Befunde wird diskutiert.

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Electronmicroscopic studies on the lipid storage in the renal tubules of Drosophila melanogaster

Summary In the cells of the renal tubules (Malpighian tubules) of Drosophila melanogaster lipids are stored in the same way as 3-hydroxykynurenin. These substances are found in dilatations of the endoplasmic reticulum. In later larval stages the lipid droplets gradually disappear. In theses stages the lipid droplets are either closely associated with the mitochondria or they are removed by concentric membrane arrays of the endoplasmic reticulum. The functional significance of these findings is discussed.

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Diese Untersuchung wurde mit Unterstützung der Deutschen Forschungsgemeinschaft durchgeführt.     

Changes in carbohydrate composition and trehalase-activity during the budding cycle of Saccharomyces cerevisiae

Summary The carbohydrate composition and the specific activity of the trehalase of cyclic partially synchronised yeast populations have been investigated. Under glucose limitation and appropriate cultural conditions synchronous growth in a chemostat was achieved. The cells accumulated the reserve carbohydrates during the single cell phase between two buddings. The rapid degradation of part of these reserves began shortly before the swelling of the bud. The importance of the mobilisation of endogenous reserves for the development of the cell is discussed.The specific activity of the trehalase changed during the budding cycle. The result gives rise to the assumption that the synthesis of this enzyme is linked to the growth cycle.     

Semisynthesis of Arg15, Glu15, Met15, and Nle15-aprotinin involving enzymatic peptide bond resynthesis

The semisynthesis of homologues of aprotinin, the bovine pancreatic trypsin inhibitor, is described. The P₁ lysine¹⁵ residue was replaced by two methods. The first procedure, which consisted of two enzymatic steps for the incorporation of other amino acids has previously been described. The second approach consisted of six steps of both enzymatic and chemical nature. The modified inhibitor, in which the lysine¹⁵-alanine¹⁶ peptide bond is hydrolyzed, was used as the starting material. All carboxyl groups of the modified inhibitor were esterified with methanol; the lysine¹⁵ methylester group was then selectively hydrolyzed. Afterward, lysine¹⁵ itself was split off. Arginine, glutamic acid, methionine, andl-2-aminohexanoic acid (norleucine, Nle) were incorporated using water-soluble carbodiimide combined with an acylation catalyst. The methylester group was used to prevent polymerization. The reactive-site peptide bonds were resynthesized using either chymotrypsin or trypsin.