Amalia Guzdek

Comparative properties of human α-1-proteinase inhibitor glycosylation variants

Variant forms of human α‐1‐proteinase inhibitor (α‐1‐PI), obtained by the treatment of human Hep G2 cells with specific inhibitors of glycosylation were tested for both inhibitory activity and heat stability. All were found to have the same second‐order association rate with human neutrophil elastase, indicating a lack of importance of the carbohydrate moiety. In contrast, incompletely glycosylated forms of α‐1‐PI were found to be heat sensitive relative to the mature protein, suggesting a role for carbohydrate in protein stabilization.

Cytokine production in human and rat macrophages and dicatechol rooperol and esters

The ability of dicatechol rooperol and esters to inhibit the production of cytokines in endotoxin-stimulated human alveolar macrophages, human blood monocyte/macrophages, histiocytic cell line U937, and rat alveolar macrophages was examined in vitro. Rooperol derivatives inhibited the production of tumour necrosis factor-alpha, interleukin-1 beta and interleukin-6. Of the esters tested on human cells, rooperol diacetate and tetraacetate were more potent inhibitors of cytokine production (IC50 in the range of 10-20 microM) than rooperol disulphate (IC50 in the range of 25-75 microM). The acetate esters also inhibited cytokine production in rat alveolar macrophages, whereas the sulphate had little effect. Rooperol and acetate esters, in the same concentration range, decreased the production of nitric oxide by rat alveolar macrophages stimulated by endotoxin. These concentrations of rooperol had no effect on cell viability, as indicated by incorporation of 14C-labelled leucine into macrophage proteins and their content of lactate dehydrogenase. The results obtained suggest that rooperol esters are potentially useful antiinflammatory agents.

Hepatocyte growth factor and retinoic acid exert opposite effects on synthesis of type 1 and type 2 acute phase proteins in rat hepatoma cells

Rat hepatoma cells H-35 cultured in serum-free medium were exposed to interleukin-6 (IL-6), interleukin-1 (IL-1), hepatocyte growth factor (HGF), retinoic acid (RA), or a mixture of these factors. Production of acute phase proteins, responding to IL-6 alone (type 2) or to the mixture of IL-6 and IL-1, was assessed by electroimmunoassay and the corresponding mRNAs were compared by Northern blot analysis. HGF enhanced IL-6-induced synthesis of alpha-2-macroglobulin but reduced synthesis of C3 complement and alpha-1-acid glycoprotein. Retinoic acid reduced the response to IL-6 of alpha-2-macroglobulin but enhanced that of alpha-1-acid glycoprotein and especially of C3 complement. In general, changes in protein secretion were correlated with the contents of their corresponding cellular mRNAs. These results indicate that hepatocyte growth factor can enhance basal or IL-6-induced gene expression of type 2 and reduce the expression of type 1 acute phase proteins, whereas the action of retinoic acid is opposite. The modulation of acute phase response by HGF and RA likely involves transcriptional factors and regulatory sequences in the genes coding for these two types of acute phase proteins.

Rooperol, an inhibitor of cytokine synthesis, decreases the respiratory burst in human and rat leukocytes and macrophages

Luminol-enhanced chemiluminescence was measured in fresh whole human blood, or human neutrophils isolated from heparinized blood, human alveolar macrophages and rat alveolar macrophages stimulated with bacterial endotoxin (LPS). Tetraacetate esters of rooperol, a dicatechol showing anticytokine activity, added to cells simultaneously with LPS inhibited the respiratory burst. The effective concentrations of rooperol were in the range of 1-10 μM depending on cell type and corresponded well with inhibition of nitric oxide production by rat alveolar macrophages. Thus rooperol may reduce some effects of excessive phagocytic activity and inflammatory reaction but by quenching free radicals production may also diminish the resistance to bacterial infections.

Rooperol tetraacetate decreases cytokine mRNA levels and binding capacity of transcription factors in U937 cells

We have previously described inhibition of the synthesis of three acute-phase inflammatory cytokines in human and rat macrophages by acetate esters of rooperol, a dicatechol of plant origin. Analysing the mechanism of anticytokine activity of rooperol, we compared levels of TNFalpha, IL-1beta and IL-6 mRNAs in the human promonocytic U937 cell line pretreated with phorbol myristate acetate (PMA) and incubated with rooperol tetraacetate (RTA) alone or in combination with LPS (500 ng/ml). It was found that 10 microM RTA decreased the levels of cytokine mRNAs both in the presence and absence of LPS, suggesting pretranslational inhibition of cytokine synthesis. Electrophoretic mobility shift analysis (EMSA) showed that RTA may influence cytokine mRNA expression by decreasing the binding activity of transcription factors NF-kappaB and AP-1.

Alpha 1 acid glycoprotein in streptozotocin diabetic rats. Some aspects of sugar moiety structure and metabolism.

SummaryThe pure alpha1 acid glycoprotein (AGP) preparation from streptozotocin diabetic rat sera showed diminished sialic acid content and lower ratios of galactose to mannose and galactose to fucose.In vivo incorporation of14C-leucine and3H-galactose into AGP of control and diabetic animals was studied 30, 60 and 120 min after administration of the radioisotopic precursors. The changed14C/3H ratio in AGP of diabetic rats suggested disturbed glycosylation of AGP in this disease. Similar activity of UDP-galactose 4-epimerase in control and diabetic rat liver was found suggesting that this enzyme has no influence on the decreased level of serum protein bound galactose.

Free and protein-bound rat serum peptides

Some of the free serum peptides circulate in blood bound to the serum proteins. Conditions used in protein precipitation influence the peptide affinity to the protein. The lability of the bonds between peptides and their carrier proteins may be of physiological importance.

In vivo and in vitro synthesis of some serum peptides

Abstract 1. 1. A high amount of [14C]leucine is incorporated in vivo into rat serum peptides after 1 hr of isotope administration. 2. 2. In vitro [14C]leucine appears in reconstituted blood plasma and in rat liver peptide mixtures. After 4hr of perfusion the radioactivities amount to 2078 ± 913 and 2120 ± 549 cpm/mg of peptides, respectively. 3. 3. The serum and liver peptides analysed show a great aggregation ability and so it is difficult to decide which of the liver peptides are destined for the serum.