Trigger factor and DnaK cooperate in folding of newly synthesized proteins.

The role of molecular chaperones in assisting the folding of newly synthesized proteins in the cytosol is poorly understood. In Escherichia coli, GroEL assists folding of only a minority of proteins and the Hsp70 homologue DnaK is not essential for protein folding or cell viability at intermediate growth temperatures. The major protein associated with nascent polypeptides is ribosome-bound trigger factor, which displays chaperone and prolyl isomerase activities in vitro. Here we show that delta tig::kan mutants lacking trigger factor have no defects in growth or protein folding. However, combined delta tig::kan and delta dnaK mutations cause synthetic lethality. Depletion of DnaK in the delta tig::kan mutant results in massive aggregation of cytosolic proteins. In delta tig::kan cells, an increased amount of newly synthesized proteins associated transiently with DnaK. These findings show in vivo activity for a ribosome-associated chaperone, trigger factor, in general protein folding, and functional cooperation of this protein with a cytosolic Hsp70. Trigger factor and DnaK cooperate to promote proper folding of a variety of E. coli proteins, but neither is essential for folding and viability at intermediate growth temperatures.

Role of region C in regulation of the heat shock gene-specific sigma factor of Escherichia coli, sigma32.

Expression of heat shock genes is controlled in Escherichia coli by the antagonistic action of the sigma32 subunit of RNA polymerase and the DnaK chaperone system, which inactivates sigma32 by stress-dependent association and mediates sigma32 degradation by the FtsH protease. A stretch of 23 residues (R122 to Q144) conserved among sigma32 homologs, termed region C, was proposed to play a role in sigma32 degradation, and peptide analysis identified two potential DnaK binding sites central and peripheral to region C. Region C is thus a prime candidate for mediating stress control of sigma32, a hypothesis that we tested in the present study. A peptide comprising the central DnaK binding site was an excellent substrate for FtsH, while a peptide comprising the peripheral DnaK binding site was a poor substrate. Replacement of a single hydrophobic residue in each DnaK binding site by negatively charged residues (I123D and F137E) strongly decreased the binding of the peptides to DnaK and the degradation by FtsH. However, introduction of these and additional region C alterations into the sigma32 protein did not affect sigma32 degradation in vivo and in vitro or DnaK binding in vitro. These findings do not support a role for region C in sigma32 control by DnaK and FtsH. Instead, the sigma32 mutants had reduced affinities for RNA polymerase and decreased transcriptional activities in vitro and in vivo. Furthermore, cysteines inserted into region C allowed cysteine-specific cross-linking of sigma32 to RNA polymerase. Region C thus confers on sigma32 a competitive advantage over other sigma factors to bind RNA polymerase and thereby contributes to the rapidity of the heat shock response.

Nitric oxide production by cells isolated from regenerating rat liver.

Nitric oxide (NO) production by cells of the regenerating liver was estimated from the amount of nitrite accumulated during 24 h in the culture media of hepatocytes, Kupffer cells and sinusoidal endothelial cells isolated at different times after partial hepatectomy (PHE). The time course of NO production was compared with the course of the proliferating activity of the same cells. During the time when liver cells pass through their first cell cycles, hepatocytes were the main producers of NO in the liver. The time-dependent changes of their NO production corresponded to those obtained with the whole liver and were inversely correlated with the DNA-synthesizing activity. The NO production by Kupffer and endothelial cells followed that by hepatocytes in this order; the time displacement between them corresponded to the schedule of their proliferating activity. The NO synthesis in non-parenchymal cells fluctuated in a similar way as in parenchymal cells and was minimal when DNA synthesis was manifest.

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 organism's 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.

Regulation of tumor necrosis factor-alpha-mRNA synthesis and distribution of tumor necrosis factor-alpha-mRNA synthesizing cells in rat liver during experimental endotoxemia.

Stimulated liver macrophages (Kupffer cells) are known to release a variety of inflammation-related substances, e.g. cytokines, prostanoids, and reactive oxygen intermediates. For instance, exposure of Kupffer cells in vitro to lipopolysaccharide (endotoxin) leads to a strongly enhanced synthesis of the mRNA for tumor necrosis factor-alpha, the release of the mature protein into culture media. These events are influenced by prostanoids and corticoid hormones. Kupffer cells are thought to be the only source of tumor necrosis factor-alpha within the hepatic sinusoid, but neither this cell specificity nor the regulatory influence of glucocorticoids or prostanoids has been confirmed in the intact organ. Using non-radioactive in situ hybridization, it was possible to obtain specific signals for tumor necrosis factor-alpha-mRNA in individual Kupffer cells uniformly distributed (as compared to Kupffer cells detected by immunohistochemistry) throughout the liver. Kupffer cells were the only cells in the hepatic sinusoids of lipopolysaccharide-perfused livers to express mRNA for tumor necrosis factor-alpha. Simultaneous addition of endotoxin plus dexamethasone and endotoxin and prostaglandin E2 completely suppressed the synthesis of this mRNA. Unexpectedly, the presence of mRNA for tumor necrosis factor-alpha was also detected in the intrahepatic bile duct epithelium of lipopolysaccharide-perfused livers. It is known that biologically active endotoxin is secreted via the bile ducts. These results seem to indicate that bile duct epithelium responds to inflammatory agents with synthesis of tumor necrosis factor-alpha-mRNA. One must also consider new functional aspects of bile duct epithelium in chronic inflammatory diseases, e.g. primary biliary cirrhosis, chronic sclerosing cholangitis or graft-versus-host disease.

Production of tumor necrosis factor-alpha, interleukin-1 and interleukin-6 in the perfused rat liver.

The kinetics of the production and release of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and interleukin-6 (IL-6) were investigated in the perfused rat liver and in primary cultures of Kupffer cells after stimulation with lipopolysaccharide (LPS). A small and transient accumulation of TNF-alpha could be detected immunohistochemically and by cytotoxicity assay in the intracellular space about 1 h after addition of LPS to the cultured cells. TNF-alpha release in the perfused liver followed similar kinetics as those found in the serum of LPS-treated rats and in primary cultures of rat Kupffer cells. The cytotoxic TNF-alpha activity of the perfusate attained its maximum (11.5 +/- 2.6 U/ml) 90 min after LPS stimulation and remained nearly constant for further 150 min. 2 microM dexamethasone reduced the production of TNF-alpha by 10 g of liver during 240 min from 46 to 16 x 10(3) units. The production of IL-1 and IL-6 by 10 g of liver during the initial 240 min was 3 and 530 x 10(3) IU, respectively. The maximal concentrations of IL-1 (1.4 +/- 0.7 IU/ml) and IL-6 (157 +/- 60 IU/ml) were found 240 min after LPS addition. The production of IL-1 was totally suppressed by 2 microM dexamethasone.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and quantitative analysis of nucleic acids by anion-exchange high-performance liquid chromatography.

A novel and reliable high-performance liquid chromatography (HPLC) method is described for the purification and quantification of double-stranded DNA. The nucleic acids may be obtained by polymerase chain reaction (PCR) or as restriction fragments from enzymatic cleavage; the separated products are devoid of contaminating material like agarose, ethidium bromide or non-specific DNA sequences. Because of the non-destructive nature of this HPLC procedure, the purified DNA is optimally suited for cloning experiments. The DNA separation by HPLC has major advantages when combined with reverse transcription (RT)-PCR. This is exemplified by analysis of the TNF-alpha mRNA obtained from endotoxin-elicited rat liver macrophages. If the standard procedure of Northern blotting is compared with the combination of RT-PCR and quantification of the PCR products by HPLC, it is obvious that the dynamic changes of tumor necrosis factor (TNF)-alpha mRNA synthesis are at least as precisely reflected with the RT-PCR/HPLC combination. The latter method is presented as a reliable and powerful tool for quantitative studies on gene expression.

Prostaglandin D2 and E2 syntheses in rat Kupffer cells are antagonistically regulated by lipopolysaccharide and phorbol ester.

Prostaglandin-synthesizing activities were demonstrated in cell-free extracts of rat Kupffer cells and characterized. The enzymatic properties of PGH2 synthase were found to be similar to those of synthases present in other organs or cell types. The specific activity of the enzyme was not changed by substances that stimulate prostanoid release by intact Kupffer cells; however, it was reduced by pretreatment of the cells with glucocorticoid hormones. On the other hand, the activities of PGD2 and PGE2 synthase were influenced differently by the kind of cell stimulation. While pretreatment of the intact cells with endotoxin and/or inhibition of protein kinase C led to an enhanced PGE2 formation in cell-free extracts, exposure to agents that enhance protein kinase C-dependent signalling pathways, e.g. phagocytotic stimuli or phorbol ester, suppressed PGE2 synthase activity and, therefore, led to enhanced PGD2 synthesis. It is in line with this observation that in vitro activation of protein kinase C of Kupffer cells resulted in a reduced PGE2 and an enhanced PGD2 synthase activity.

Protein kinase C involved in zymosan-induced release of arachidonic acid and superoxide but not in calcium ionophore-elicited arachidonic acid release or formation of prostaglandin E2 from added arachidonate.

Zymosan and phorbol ester induced in liver macrophages the release of arachidonic acid, prostaglandin E2, and superoxide; the calcium ionophore A 23187 elicited a release of arachidonic acid and prostaglandin E2 but not of superoxide, and exogenously added arachidonic acid led to the formation of prostaglandin E2 only. The zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide was dose-dependently inhibited by staurosporine and K252a, two inhibitors of protein kinase C, and by pretreatment of the cells with phorbol ester which desensitized protein kinase C. The release of arachidonic acid or prostaglandin E2 following the addition of A 23187 or arachidonic acid was not affected by these treatments. Zymosan and phorbol ester but not A 23187 or arachidonic acid induced a translocation of protein kinase C from the cytosol to membranes in intact cells. These results demonstrate an involvement of protein kinase C in the zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide; the release of arachidonic acid and prostaglandin E2 elicited by A 23187 and the formation of prostaglandin E2 from exogenously added arachidonic acid, however, is independent of an activation of protein kinase C.

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.

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.

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.

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.

Effect of fluoride, pertussis and cholera toxin on the release of arachidonic acid and the formation of prostaglandin E2, D2, superoxide and inositol phosphates in rat liver macrophages.

Fluoride elicited in liver macrophages a release of arachidonic acid and prostaglandins but not formation of inositol phosphates or superoxide. The effects of fluoride required extracellular calcium and were inhibited by staurosporine and by phorbol ester treatment of the cells. Furthermore, fluoride led to a translocation of protein kinase C from the cytosol to membranes. This indicates that the calcium-dependent protein kinase C is involved in the action of fluoride. Cholera toxin decreased the zymosan-induced release of arachidonic acid and prostaglandins but not of inositol phosphates or superoxide. Pertussis toxin ADP-ribosylated a 41,000 molecular weight membrane protein; enhanced specifically the zymosan-induced formation of prostaglandin(PG)E2 but did not affect the zymosan-induced release of arachidonic acid, PGD2, inositol phosphates or superoxide. These data suggest that activation of phospholipase (PL)A2, phosphoinositide (PI)-specific PLC and NADPH oxidase in liver macrophages is most probably not mediated by activation of guanine nucleotide binding (G)-proteins coupled directly to these enzymes.

Activation of phospholipase C is not correlated to the formation of prostaglandins and superoxide in cultured rat liver macrophages.

This study evaluates the role of inositol phosphates as possible mediators of the activation of phospholipase A2 and NADPH oxidase in cultured rat liver macrophages. Inositol phosphate formation was achieved by zymosan, immune complexes, latex particles and calcium ionophore while the release of arachidonic acid and the formation of prostaglandin E2 was also elicited by phorbol ester and NaF, but not by latex particles; generation of superoxide was obtained by zymosan and phorbol ester only. The kinetics of the formation of inositol phosphates revealed that within the first few minutes after zymosan addition inositol trisphosphate was formed, followed by inositol bisphosphate and inositol monophosphate. Pre-treatment of the cells with dexamethasone or removal of extracellular calcium led to an inhibition of the zymosan-induced formation of inositol phosphates and prostaglandin E2 but had no effect on the generation of superoxide; inhibition of the Na+/H+ exchanger by removal of extracellular sodium ions led to a decrease of the zymosan-induced synthesis of prostaglandin E2, but did not affect the formation of inositol phosphates and superoxide. Pre-treatment of the cells with phorbol ester decreased the zymosan-induced synthesis of prostaglandin E2 and superoxide, but even enhanced the zymosan-induced formation of inositol phosphates. These data indicate that in cultured rat liver macrophages the formation of prostaglandins and superoxide cannot be correlated to an activation of phospholipase C.

Arachidonate metabolism in macrophages is affected by albumin.

The incorporation of externally added arachidonic acid into membrane lipids of cultured liver macrophages was found to be strongly inhibited by albumin. Furthermore this protein increased the appearance of radiolabelled arachidonic acid in cell media from [3H] arachidonic acid-prelabelled cells but inhibited almost totally the stimulus-induced formation of prostanoids. Analysis of the membrane lipids revealed that in the presence of albumin arachidonic acid was released to almost the same percentage from all phospholipids, independently of the stimulus. In contrast, in the absence of albumin a significant loss of arachidonic acid was observed for phosphatidylcholine and phosphatidyl-inositol only and the degree of the decrease in label was dependent on the stimulus. Although the exact mechanisms for these actions of albumin are not yet known this protein clearly exerts an inhibitory effect on arachidonic acid incorporation into membrane lipids as well as on the synthesis of different eicosanoids in macrophages; on the other hand albumin leads to an enhanced appearance of arachidonic acid in the cell media. Therefore this protein may be able to control the level of free arachidonic acid within and outside the cell and thereby the conversion of arachidonic acid into eicosanoids.

Fc receptors mediate prostaglandin and superoxide synthesis in cultured rat Kupffer cells.

Latex beads with covalently bound bovine serum albumin were prepared and coated with anti-BSA immunoglobulin G. These particles were shown to possess on their surfaces a defined quantity of the antibody with the Fc portions exposed to the medium. One homologous and two heterologous antibodies of the G class were used and compared in terms of their binding to the rat Kupffer cells and their ability to elicit the typical phagocytotic responses. These particles were phagocytosed by rat Kupffer cells and elicited synthesis of prostaglandins and superoxide anion radicals. A significant release of superoxide into the medium was observed in the presence of cytochalasin B only. The data presented here suggest that a) Fc-carrying particles can be bound to Kupffer cells and elicit responses via specific receptors; b) coating with the homologous antibody yields the most effective particles; c) superoxide release into the surrounding medium is most abundant when the particle-binding membrane areas are prevented from forming phagocytotic vesicles.

4-Bromophenacyl bromide inhibits prostaglandin D2 synthesis from arachidonic acid rather than phospholipase A2 activity in liver macrophages.

4-Bromophenacyl bromide at a concentration of 50 microM does not inhibit phospholipase A2 activity in liver macrophages. Rather, this compound increases the amount of radioactivity released from [3H]arachidonate-prelabeled Kupffer cells and leads to the formation of small amounts of thromboxane, prostaglandin D2 and prostaglandin E2. Also the zymosan-induced formation of thromboxane and prostaglandin E2 from endogenous sources which is thought to involve phospholipase A2 remains unaffected in the presence of this compound. The generation of superoxide and the formation of prostaglandin D2 from arachidonate and after stimulation of the cells with zymosan, however, are blocked by 4-bromophenacyl bromide. Furthermore, this compound suppresses the incorporation of externally added arachidonate into membrane lipids of the cells. 4-Bromophenacyl bromide seems, therefore, not to be a useful tool to demonstrate the involvement of phospholipase A2 in complex biological systems.

Independent regulation of thromboxane and prostaglandin synthesis in liver macrophages.

Incubation of liver macrophages with zymosan, phorbol ester and calcium ionophore A 23187 led to the formation of thromboxane, prostaglandin E2 and prostaglandin D2, whereas after external addition of arachidonic acid prostaglandin E2 and prostaglandin D2 only were found. This was confirmed by the use of labeled arachidonic acid given together with the stimuli. When the liver macrophages were prelabeled with [3H]arachidonic acid, and zymosan and [14C]arachidonic acid were added simultaneously, [3H]-label only was found in thromboxane whereas both [3H]- and [14C]-labeled PGE2 and PGD2 were detected in the cell medium. These data suggest that in cultured rat liver macrophages externally added arachidonic acid is accessible to the cyclooxygenase supplying prostaglandin H2 for prostaglandin E2 and D2 synthesis but not for thromboxane synthesis.

Ca2+ requirement of prostanoid but not of superoxide production by rat Kupffer cells.

The release of the prostanoids prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+ to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half-maximal rate of PGE2 release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6-0.7 microM free extracellular Ca2+ and greater than or equal to 100 microM free Ca2+ was required to stimulate PGE2 formation maximally. The calmodulin antagonist R24571 partially inhibited the release of PGE2 elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE2 irrespective of the stimulus. TMB 8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE2 induced by zymosan and phorbol ester. The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+-dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+ and mobilization of Ca2+ from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.