Hanspeter Henninger

Rat hepatic sinusoidal endothelial cells in monolayer culture: Biochemical and ultrastructural characteristics

Sinusoidal endothelial cells were isolated by collagenase-pronase digestion of rat livers followed by centrifugal elutriation. The main endothelial cell fraction consisted of more than 85% endothelial cells as shown by electron microscopy and enzyme histochemistry. Contamination by Kupffer cells was less than 5%. The endothelial cells formed a coherent stable monolayer on dishes coated with collagen type IV in the presence of an RPMI 1640 medium supplemented with 4% Ultroser. Fc receptors were undetectable immediately after elutriation but reappeared after 12 h in culture. Von Willebrand factor (formerly factor VIII-related antigen) could not be detected unequivocally by immunofluorescence. Unchallenged endothelial cells did not produce eicosanoids. In the presence of free arachidonate, however, prostaglandins D2 and E2 as well as thromboxane B2 and 6-keto-prostaglandin F1 alpha were detected by radioimmunoassay and by high-performance liquid chromatography analysis of [3H]arachidonate-exposed cells. Cells treated with the Ca2+ ionophore A23187 produced the same spectrum of immunologically measured prostanoids. In contrast to Kupffer cells in primary culture, eicosanoid formation by endothelial cells was neither triggered by phagocytotic stimuli nor suppressed by pretreatment with dexamethasone.

Studies on synthesis and degradation of eicosanoids by rat hepatocytes in primary culture

The potential of hepatocytes in primary cultures to degrade the prostanoids produced by Kupffer cells and to synthesize eicosanoids, especially leukotriene B4, after treatment with D-galactosamine was studied. Hepatocytes in primary cultures showed a substantial capability to degrade all the prostanoids produced by stimulated Kupffer cells. The rate of degradation, approx. 2 pmol/min per 10(6) hepatocytes, was nearly the same for the prostaglandins D2, E2 and F2a. Lower rates were determined for thromboxane B2 (0.4 pmol/min per 10(6) cells) and for 6-ketoprostaglandin F1a (0.2 pmol/min per 10(6) cells). The degradation products of these prostanoids lacked biological activity, e.g., reactivity with specific antibodies and the ability to contract segments of rabbit femoral artery. In the presence of 30 microM arachidonic acid, hepatocytes produced only very small amounts of prostaglandins and thromboxane, ranging from less than or equal to 22 to 50 fmol/30 min per 10(6) cells. Neither untreated nor D-galactosamine-treated hepatocytes released significant amounts of leukotriene B4. Hepatocytes appear to be the site of degradation rather than synthesis of eicosanoids in the liver.

A pAO1-encoded molybdopterin cofactor gene (moaA) of Arthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein

A gene homologous tomoaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor ofEscherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 ofArthrobacter nicotinovorans. The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880. The pAO1-encodedmoaA gene fromA. nicotinovorans was expressed inE. coli as an active protein that functionally complementedmoaA mutants. Its reduced amino acid sequence shows 43% identity to theE. coli MoaA, 44% to the NarAB gene product fromBacillus subtilis, and 42% to the gene product of two contiguous ORFs fromMethanobacterium formicicum. N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins. This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in thefixZ gene product fromRhizobium leguminosarum. Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement themoaA mutation inE. coli. A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins. These two Cys-rich sequences may be involved in the coordination of a metal ions. The pAO1 copy ofmoaA may not be unique in theA. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.

Regulation of interleukin-6 receptor expression in rat Kupffer cells: modulation by cytokines, dexamethasone and prostaglandin E2

Interleukin-6 has a variety of biological effects, mainly on the immune system. The regulation of this signal at both the site of production and the site of action is necessary to maintain the organisms homeostasis. In the microenvironment of the hepatic sinusoids, Kupffer cells as resident macrophages are the most potent source of interleukin-6 during inflammation. This cytokine is an important signal to hepatocytes during the early stages of the acute-phase response, leading to the expression of several major plasma proteins. Kupffer cells were found to express interleukin-6 receptor constitutively. Interleukin-6 decreased the level of interleukin-6 receptor mRNA, indicating an autocrine pathway by which Kupffer cells regulate their responsiveness to interleukin-6. Furthermore, lipopolysaccharide, tumor necrosis factor-alpha, interferon-gamma, interleukin-1 beta and phorbol ester induced interleukin-6 production and, at the same time, suppressed the level of interleukin-6 receptor mRNA. The existence of an autocrine loop in rat Kupffer cells may be physiologically relevant, as it would contribute to a regulated interleukin-6 signal chain in the liver. The anti-inflammatory mediators dexamethasone or PGE2 and its second messenger, cyclic AMP, increased interleukin-6 receptor mRNA, whereas prostaglandin D2 or the Ca2+ ionophore, A 23187, were without effect. The changes in interleukin-6 mRNA were paralleled by the number of interleukin-6 receptors present on Kupffer cells as detected by binding of 125I-interleukin-6. These results suggest the existence of control mechanisms involving several soluble mediators that help balance the level of interleukin-6-R mRNA in rat liver macrophages.

The design of an alternative, covalently flavinylated 6-hydroxy-d-nicotine oxidase by replacing the FAD-binding histidine by cysteine and reconstitution of the holoenzyme with 8-(methylsulfonyl)FAD

The cofactor of several flavoenzymes is autocatalytically bound to the polypeptide via a histidyl(N3)‐(8α)‐FAD linkage which makes the generation of apoenzyme difficult. We introduced an alternative covalent protein‐FAD bond at the active site of 6‐hydroxy‐n‐nicotine oxidase (6HDNO) by replacing the FAD‐binding histidine with cysteine. The resulting mutant enzyme was expressed with noncovalently attached cofactor. Incubation with 8‐(methylsulfonyl)FAD, and less efficiently with 8‐chloro‐FAD, resulted in the spontaneous replacement of the noncovalently bound FAD by the flavin derivative and the formation of an 8‐(N‐acetylcysteinyl)FAD linkage. The flavinylated 6HDNO.cys exhibited close to wild‐type activity levels. This strategy may be generally applicable to the attachment of artificially designed flavin derivatives to the active site of covalently flavinylated enzymes.

Depletion of cytidine triphosphate as a consequence of cellular uridine triphosphate deficiency

The synthesis of CTP from UTP, catalyzed by CTP synthetase (EC 6.3.4.2), would imply that severe UTP deficiency causes a reduction of cellular CTP synthesis followed by a decrease in CTP pools. By contrast, UTP depletion induced by D-galactosamine in rat liver in vivo has been shown to be associated with a 2-fold increase in the hepatic content of CTP [ 1,2]. This result is confirmed in the present communication by means of high-pressure liquid chromatography of hepatic nucleotides. In vivo, the cytidine present in plasma [3] can contribute significantly to the synthesis of CTP on the salvage pathway. We have therefore studied hepatoma cell suspensions in vitro in order to evaluate the influence of the cellular level of UTP on CTP pools in the absence of extracellular cytidine. Under this condition a close correlation over a wide range was demonstrated between UTP and CTP contents. This correlation was independent from the agents used to induce UTP deficiency. These included inhibitors of de novo pyrimidine nucleotide synthesis and the uridylate-trapping amino sugars D-glucosamine (GlcN) and D-galactosamine (GalN). The analysis of pyrimidine nucleotides by highpressure liquid chromatography has shown an excellent agreement with our earlier enzymatic measurements ]4,51.