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Biomedical subjects

K Decker

Publications and source records attributed to K Decker.

At least 73 records · Page 4Linked to original sources
PubMed1992

How and why are some riboflavin coenzymes covalently attached to proteins?

Covalent flavinylation as elucidated by the formation of the histidyl(N3)-8 alpha-flavin bond in 6-hydroxy-D-nicotine oxidase of A. oxidans proceeds by an non-enzymatic mechanism. Incubation of the apoenzyme, FAD and a three-carbon phosphate ester at neutral pH leads to the formation of an enzymatically fully active holoenzyme. The role of His71 in this process was illustrated by site-directed mutagenesis. Nevertheless, the question whether covalent attachment of the cofactor in a holoenzyme has prevailed throughout the evolutionary screening process because of biological significance or whether it is a chance event of neutral selective value is still open.

Animals↗
PubMed1991

Ca(2+)-induced reversible translocation of phospholipase A2 between the cytosol and the membrane fraction of rat liver macrophages.

In cell-free extracts of rat liver macrophages (Kupffer cells) phospholipase A2 was found to be rapidly associated with the particulate fraction in a Ca(2+)-dependent manner at Ca2+ concentrations of 0.1-1.0 microM. This is also the range of the levels of intracellular Ca2+ reported for basal and various stimulated conditions. After translocation, phospholipase A2 could be released from the membranes in the presence of Ca2+ chelators, increasing the specific activity of phospholipase A2 in the supernatant fraction. These findings support the view that translocation is a regulatory mechanism of phospholipase A2 by bringing the enzyme to its substrate. Unlike the situation with protein kinase C, Mg2+ exerted little effect on phospholipase A2 translocation, indicating that this process is regulated in vivo mainly by fluctuations of the intracellular Ca2+ content.

Animals↗
PubMed1991

Synergistic effect of magnesium and calcium ions in the activation of phospholipase A2 of liver macrophages.

In cell-free extracts of rat liver macrophages (Kupffer cells) phospholipase A2 was found to be strongly activated at free Ca2+ concentrations from 100 nM to 1 microM in the presence of 4 mM free Mg2+. This is within the range of intracellular free Ca2+ reported for basal and various stimulated conditions, respectively. Ca2+ alone increased phospholipase A2 activity at high Ca2+ concentrations (1 mM) whereas Mg2+ alone had only little stimulatory effect. Calmodulin does not seem to participate in the regulation of phospholipase A2 although it relieved the inhibition of phospholipase A2 activity by calmodulin antagonists.

Animals↗
PubMed1991

Regulation of nitric oxide production by stimulated rat Kupffer cells.

Macrophages have been described to release nitric oxide (NO) as a cytotoxic radical. This highly unstable substance is as well known as endothelium-derived relaxing factor produced by vascular endothelial cells. Because of its cytotoxic activity the synthesis of NO by rat Kupffer cells, the liver macrophages, upon stimulation with endotoxin (lipopolysaccharide; LPS) and tumor necrosis factor-alpha (TNF-alpha) in combination with prostaglandin E2 (PGE2) and dibutyryl cAMP (dBcAMP) was studied. Kupffer cells were stimulated after 48 h of primary culture. NO was quantified as NO2- in the cell medium 24 h after stimulation. LPS stimulated NO generation 5- to 10-fold over the basal level. This increase could be further enhanced by PGE2 and dBcAMP, especially when added 1 h after LPS. NO generation after stimulation with LPS or LPS + PGE2 depended on the simultaneous production of PGE2 by the stimulated Kupffer cells. It could be partly inhibited by anti-PGE2 antibody or acetylsalicylic acid. While murine TNF-alpha did not stimulate NO synthesis significantly, added PGE2 raised NO synthesis about 6-fold. The addition of dBcAMP to TNF-alpha in the same concentration as with LPS, however, had no effect. Thus, stimulation by LPS + PGE2 equals that of LPS + dBcAMP whereas TNF-alpha + PGE2 does not equal TNF-alpha + dBcAMP, indicating differences in the mode of action of PGE2 on LPS- or TNF-alpha-treated Kupffer cells.

Animals↗
PubMed1991

Endotoxin-refractory liver macrophages secrete tumor necrosis factor-alpha upon viral infection.

Rat liver macrophages (Kupffer cells) secrete tumor necrosis factor-alpha (cachectin) after exposure to Newcastle disease virus or bacterial endotoxin. Macrophages treated with endotoxin become refractory and fail to release tumor necrosis factor-alpha to a secondary challenge with endotoxin. The acquisition of the refractory state is dose-dependent, requires the continuous presence of endotoxin for a minimum of 8 h, is transient, and reversible. Endotoxin, however, renders Kupffer cells unresponsive only to itself. When endotoxin-refractory macrophages are activated by Newcastle disease virus, they still secrete tumor necrosis factor-alpha in amounts expected with this stimulus. Immunoprecipitation studies show that the precursor of tumor necrosis factor-alpha is found only in lysates of endotoxin-sensitive, but not in refractory macrophages, thus arguing against a post-translational regulatory process. Whereas prostaglandin E2 inhibits the production of tumor necrosis factor-alpha in response to endotoxin and viruses, it does not appear to mediate the refractory state.

Animals↗
PubMed1991

Signal transduction in endotoxin-stimulated synthesis of TNF-alpha and prostaglandin E2 by rat Kupffer cells. Role of extracellular calcium ions and protein kinase C.

Endotoxin is a well established elicitor of cytokine production in mononuclear cells. Nevertheless, the path of signal transduction between the crucial contact of the cells with endotoxin (lipopolysaccharide) and the synthesis and release of the mediators is yet poorly understood. In particular, the involvement of Ca2+ and protein kinase C in this process is still a matter of controversy. Here, it will be demonstrated that removal of extracellular Ca2+ by EGTA does not have a significant effect on the endotoxin-stimulated production of tumor necrosis factor-alpha (TNF-alpha) and on total protein synthesis in rat Kupffer cells. However, the release of prostaglandin E2 could not be raised above the basal level under these conditions. Treatment with inhibitors of protein kinase C such as the isoquinoline derivative, H-7, or staurosporin is without influence on TNF-alpha synthesis. The depletion of protein kinase C through preincubation of rat Kupffer cells with phorbol 12-myristate 13-acetate for 24 h was also without effect on TNF-alpha production. The effectiveness of these inhibitors under the conditions used was ascertained by measurement of the O2- release from the same cell batches. Superoxide production known as protein kinase C-dependent in Kupffer cells (Dieter et al. (1986) Eur. J. Biochem. 86, 451-457) was suppressed in a dose-dependent manner by staurosporin or after prolonged pretreatment with the phorbol ester. H-7 decreased superoxide production only slightly in high doses that severely harm the Kupffer cells. Prostaglandin E2 release, although clearly protein-kinase C-dependent in phagocytosing rat Kupffer cells, is not decreased following exposure to lipopolysaccharide in the presence of protein kinase C inhibitors.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Fate of exogenous and endogenous prostaglandins D2 and E2 in the perfused rat liver.

The degradation of radiolabelled exogenous PGD2 and PGE2 was compared to that of endogenous labelled prostaglandins synthesized after stimulation with PMA in the perfused rat liver. With exogenous PGD2 and PGE2 the same degradation products were found in the perfused liver as in hepatocyte primary cultures. The major metabolite of PGD2 was dinor-PGD2 while tetranor-PGE1 was the main degradation product of PGE2. Some polar metabolites and tritiated water were also formed. The metabolites detected with endogenous prostaglandins were similar to those obtained with exogenous PGD2. Over 99% of the labelled prostaglandins were degraded in the recirculating perfusion within 40 min. In the open perfusion system, 95% of endogenous PGD2 was calculated to be degraded after a single passage through the liver, which suggests that hepatocytes play an important role in the removal of biologically active prostaglandins.

Animals↗
PubMed1990

Activation of GD3 synthase by sex steroid hormones in cultured rat hepatocytes.

The influence of sex steroid hormones on the activities of GM3 and GD3 synthases in isolated hepatocytes was studied. Progesterone (0.1 - 2.0 microM), beta-estradiol (0.1 - 1 microM), and testosterone (0.1 - 1 microM) activate GD3 but not GM3 synthase when added directly to hepatocytes cultured in modified William's E medium.

Animals↗
PubMed1990

Sialyltransferase activities in cultured rat hepatocytes.

Previous studies on the age and sex dependency of the ganglioside patterns in rat liver in vivo and the concomitant determination of the activities of some enzymes involved in these pathways revealed the prominent role of the sialylation of GM3 to GD3 in determining the flow to the mono (a)- and polysialo (b)-series, respectively. Here, the influence of hormones on the activities of GM3 and GD3 synthases in isolated hepatocytes was studied. The combination of several factors (insulin, glucagon, epidermal growth factor, glucocorticoids) was found to be necessary for maintaining in vivo activity levels of GD3- but not of GM3-synthase.

Animals↗
PubMed1990

Virus-vs endotoxin-induced activation of liver macrophages.

The response of liver macrophages (Kupffer cells) to distinct pathogenic material was investigated by comparing virus- and endotoxin-induced macrophage activation. Endotoxin-induced stimulation and induction with Newcastle disease virus (NDV) or Sendai virus led to the release of the same pattern of prostanoids characterized by a predominant production of prostaglandin E2 (PGE2). With respect to peptide mediators, hepatic macrophages secreted tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 after viral induction and endotoxin treatment, respectively. In response to viruses, however, much more interleukin-6 and TNF-alpha was detected than after endotoxin stimulation. Interferon type I (interferon-alpha/beta), on the other hand, was only detected in the supernatants of macrophages infected with viruses, but not of those exposed to endotoxin. This study also revealed that rat TNF-alpha exists in several soluble species, some of which are glycosylated.

Animals↗
PubMed1990

Interdependence of tumor necrosis factor, prostaglandin E2, and protein synthesis in lipopolysaccharide-exposed rat Kupffer cells.

Kupffer cells are the main producers of tumor necrosis factor-alpha (TNF; cachectin) and eicosanoids in the liver exposed to lipopolysaccharide (endotoxin; LPS). A very rapid but transient release of TNF is followed by a slow, steady synthesis of prostaglandin E2 (PGE2). TNF itself is able to provoke eicosanoid synthesis in Kupffer cells; the rate and pattern of prostaglandin production are similar to those observed after treatment with LPS. Anti-TNF antibodies completely neutralize TNF action on Kupffer cells, thus ruling out any participation of contaminating LPS. LPS stimulation of PGE2 production in Kupffer cells is reduced by the antiserum to 50%, indicating an involvement of TNF in the stimulatory action of LPS. On the other hand, PGE2, a potent inhibitor of LPS-elicited TNF release, is able to suppress LPS- but not TNF-stimulated eicosanoid synthesis in rat Kupffer cells. In addition to this autocrine circuit, extrahepatic factors participate in the regulation of Kupffer cell activation: glucocorticoids not only inhibit TNF or prostaglandin production, they also reverse the LPS-specific changes in the prostaglandin pattern of Kupffer cells. LPS, TNF or cycloheximide when given alone in the concentration range applied in this study do not affect the viability of rat Kupffer cells. However, the combinations of cycloheximide and either LPS or TNF cause rapid death of the cultured cells. The cytolytic potential of either combination cannot be alleviated by treatment with glucocorticoids.

Animals↗
PubMed1990

Ganglioside biosynthesis in rat liver: different distribution of ganglioside synthases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells.

The activities of five glycolipid-glycosyltransferases, GL2, GM3, GM2, GM1, and GD1a synthase, were determined in a cell-free system with homogenate protein of total rat liver, isolated hepatocytes, Kupffer cells, and sinusoidal endothelial cells. In rat liver parenchymal and nonparenchymal cells ganglioside synthases were distributed differently. Compared to hepatocytes, Kupffer cells expressed a nearly sevenfold greater activity of GM3 synthase, but only 14% of GM2, 19% of GM1, and 67% of GD1a synthase activity. Sinusoidal endothelial cells expressed a pattern of enzyme activities quite similar to that of Kupffer cells with the exception of higher GM2 synthase activity. Activity of GL2 synthase was distributed unifromly in parenchymal and nonparenchymal cells of rat liver, but differed by sex. It was 1 to 2 orders of magnitude below that of all the other ganglioside synthases investigated. The results indicate GL2 synthase regulates the total hepatic ganglioside content, and hepatocytes but not nonparenchymal liver cells have high enzymatic capacities to form a-series gangliosides more complex than GM3.

Animals↗
PubMed1990

Interleukin-6 release by rat liver macrophages.

Tissue macrophages of the liver (Kupffer cells) release interleukin-6 (IL-6) in vitro. Since Kupffer cells reside in close proximity to hepatocytes, which are major target cells of IL-6, the regulation of IL-6 release by hepatic macrophages has been investigated in this study. Using the hybridoma growth test to detect IL-6, we found that Kupffer cells already maximally release IL-6 at endotoxin concentrations as low as 1.0 ng/ml. The stimulated secretion of IL-6 was increased 4-8-fold by endotoxin when compared to the control macrophages incubated in serum-containing medium alone. The preincubation of macrophages with interferon-gamma enhanced the capacity of Kupffer cells to respond to endotoxin. The secretion of IL-6 could also be induced by interleukin (IL)-1 beta and tumor necrosis factor (TNF-alpha). The most potent inducers, however, were the paramyxoviruses Newcastle Disease Virus and Sendai Virus. The release of IL-6 by macrophages upon stimulation with endotoxin was almost completely inhibited by 1 microM dexamethasone. Whereas 100 nM of prostaglandin E2 (PGE2) inhibited the release of TNF-alpha in rat Kupffer cells, it did not affect the secretion of IL-6.

Animals↗
PubMed1990

Labeling of precursor pools for glycosphingolipid biosynthesis. Incorporation of [3H]galactose by rat hepatocytes in primary culture.

UDP-galactose and UDP-glucose are the immediate sources of monosaccharide residues in glycosphingolipid biosynthesis. The incorporation of [6-3H]D-galactose into these compounds was measured in primary cultures of rat hepatocytes, which take up and metabolize galactose rapidly. The UDP-glucose and UDP-galactose content of hepatocytes, determined enzymatically and by the HPLC-analysis of UDP-sugars, was 1.87 +/- 0.22 and 0.51 +/- 0.06 nmol/mg protein, respectively. Galactose concentrations in the medium of up to 7.5 microM did not influence the intracellular levels of UDP-glucose and UDP-galactose. Although the specific radioactivity of these precursor pools did not reach a constant plateau, conditions were defined that allow the calculation of rates of glycolipid synthesis from added labeled galactose. They include the replacement of glucose in the culture medium by sodium pyruvate and D-galactose.

Animals↗