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Targeting of transmembrane proteins to the endoplasmic reticulum (ER) proceeds via either the signal recognition particle (SRP) or the guided entry of tail-anchored proteins (GET) pathway, consisting of Get1 to -5 and Sgt2. While SRP cotranslationally targets membrane proteins containing one or multiple transmembrane domains, the GET pathway posttranslationally targets proteins containing a single C-terminal transmembrane domain termed the tail anchor. Here, we dissect the roles of the SRP and GET pathways in the sorting of homologous, two-membrane-spanning K(+) channel proteins termed Kcv, Kesv, and Kesv-VV. We show that Kcv is targeted to the ER cotranslationally via its N-terminal transmembrane domain, while Kesv-VV is targeted posttranslationally via its C-terminal transmembrane domain, which recruits Get4-5/Sgt2 and Get3. Unexpectedly, nascent Kcv recruited not only SRP but also the Get4-5 module of the GET pathway to ribosomes. Ribosome binding of Get4-5 was independent of Sgt2 and was strongly outcompeted by SRP. The combined data indicate a previously unrecognized cotranslational interplay between the SRP and GET pathways.

Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation.

The Hsp70 Ssb serves a dual role in de novo protein folding and ribosome biogenesis; however, the mechanism by which Ssb affects ribosome production is unclear. Here we establish that Ssb is causally linked to the regulation of ribosome biogenesis via the TORC1-Sch9 signaling pathway. Ssb is bound to Sch9 posttranslationally and required for the TORC1-dependent phosphorylation of Sch9 at T737. Also, Sch9 lacking phosphorylation at T737 displays significantly reduced kinase activity with respect to targets involved in the regulation of ribosome biogenesis. The absence of either Ssb or Sch9 causes enhanced ribosome aggregation. Particularly with respect to proper assembly of the small ribosomal subunit, SSB and SCH9 display strong positive genetic interaction. In combination, the data indicate that Ssb promotes ribosome biogenesis not only via cotranslational protein folding, but also posttranslationally via interaction with natively folded Sch9, facilitating access of the upstream kinase TORC1 to Sch9-T737., The yeast Hsp70 homolog Ssb is a chaperone that binds translating ribosomes where it is thought to function primarily by promoting nascent peptide folding. Here the authors find that the ribosome biogenesis defect associated with the loss of Ssb is attributable to a specific disruption in TORC1 signaling rather than defects in ribosomal protein folding.

Ribosome stalling is an important incident enabling the cellular quality control machinery to detect aberrant mRNA. Saccharomyces cerevisiae Hbs1-Dom34 and Ski7 are homologs of the canonical release factor eRF3-eRF1, which recognize stalled ribosomes, promote ribosome release, and induce the decay of aberrant mRNA. Polyadenylated nonstop mRNA encodes aberrant proteins containing C-terminal polylysine segments which cause ribosome stalling due to electrostatic interaction with the ribosomal exit tunnel. Here we describe a novel mechanism, termed premature translation termination, which releases C-terminally truncated translation products from ribosomes stalled on polylysine segments. Premature termination during polylysine synthesis was abolished when ribosome stalling was prevented due to the absence of the ribosomal protein Asc1. In contrast, premature termination was enhanced, when the general rate of translation elongation was lowered. The unconventional termination event was independent of Hbs1-Dom34 and Ski7, but it was dependent on eRF3. Moreover, premature termination during polylysine synthesis was strongly increased in the absence of the ribosome-bound chaperones ribosome-associated complex (RAC) and Ssb (Ssb1 and Ssb2). On the basis of the data, we suggest a model in which eRF3-eRF1 can catalyze the release of nascent polypeptides even though the ribosomal A-site contains a sense codon when the rate of translation is abnormally low.

NAC acts as a modulator of SRP function. It can bind to signal sequences directly. SRP initially displaces NAC from RNCs; however, when the signal sequence emerges, trimeric NAC·RNC·SRP complexes form. Upon docking NAC·RNC·SRP complexes to the ER, NAC remains bound, allowing NAC to shield cytosolically exposed nascent chain domains., Nascent polypeptide-associated complex (NAC) was initially found to bind to any segment of the nascent chain except signal sequences. In this way, NAC is believed to prevent mistargeting due to binding of signal recognition particle (SRP) to signalless ribosome nascent chain complexes (RNCs). Here we revisit the interplay between NAC and SRP. NAC does not affect SRP function with respect to signalless RNCs; however, NAC does affect SRP function with respect to RNCs targeted to the endoplasmic reticulum (ER). First, early recruitment of SRP to RNCs containing a signal sequence within the ribosomal tunnel is NAC dependent. Second, NAC is able to directly and tightly bind to nascent signal sequences. Third, SRP initially displaces NAC from RNCs; however, when the signal sequence emerges further, trimeric NAC·RNC·SRP complexes form. Fourth, upon docking to the ER membrane NAC remains bound to RNCs, allowing NAC to shield cytosolically exposed nascent chain domains not only before but also during cotranslational translocation. The combined data indicate a functional interplay between NAC and SRP on ER-targeted RNCs, which is based on the ability of the two complexes to bind simultaneously to distinct segments of a single nascent chain.

LPS and MTP-PE (liposome-encapsulatedN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-:[1',2'-dipalmitoyl-sni-glycero-3-(hydroxy-phosphoryl-oxyl)] etylamide) induce in liver macrophages a synthesis and release of TNF-α, nitric oxide and prostanoids. Both agents induce an expression of mRNA's encoding TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and of corresponding proteins. LPS and MTP-PE induce a rapid activation of the extracellular regulated kinase (ERK) isoenzymes-1 and -2. Inhibition of map kinase isoenzymes leads to a decreased release of TNF-α, nitric oxide and prostaglandin (PG) E2after both agents. The transcription factors NF-κB and AP-1 are strongly activated by LPS within 30 minutes. MTP-PE induces a weak activation of both transcription factors only after 5 hours. Inhibition of NF-κB inhibits the LPS- but not the MTP-PE-induced release of TNF-α, nitric oxide and PGE2. PGE2release after LPS is higher than after MTP-PE. Exogenously added PGE2inhibits the activation of map kinase and TNF-α release by LPS, but not by MTP-PE. Release of nitric oxide after LPS and MTP-PE is enhanced after prior addition of PGE2. PGD2is without any effect. MTP-PE, but not LPS, induces a cytotoxicity of Kupffer cells against P815 tumor target cells. The MTP-PE-induced cytotoxicity is reduced by TNF-α neutralizing antibodies, indicating the involvement of TNF-α. Thus our results suggest that the different potencies of LPS and MTP-PE as immunomodulators probably result from different actions on Kupffer cells, resulting in differences in the amounts and kinetics of released TNF-α and PGE2, and that PGE2plays an important regulatory role in the action of LPS, but not in the actions of MTP-PE.

Human THP-1 leukemia cells differentiate along the monocytic lineage following exposure to phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D3 (VD3). In the monocytic cell line THP-1, PMA treatment resulted in a more differentiated phenotype than VD3, according to adherence, loss of proliferation, phagocytosis of latex beads, and expression of GD11b and CD14. Both differentiating substances induced similar effects in the release of superoxide anions (O2-). VD3-differentiated cells did not release prostaglandin E2 (PGE2), in contrast to PMA-differentiated cells, and in PMA-differentiated cells phospholipase A2 (PLA2) activity and expression was increased. Lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α (TNF-α) release was higher in PMA-treated cells. PMA- but not VD3-differentiation resulted in a translocation of protein kinase C (PKC) isoenzymes to membrane fractions. Both differentiating agents up-regulated the expression of PKC isoenzymes. Whereas VD3 elevated mainly the expression of PKC-β, PMA caused a strong increase in PKC-δ and a weak increase in PKC-α, PKC-, and PKC- expression. These results indicate that phorbol ester and the active metabolite of vitamin D induce different signal pathways, which might result in different achievement of differentiation.

We have studied activation of phospholipase (PL) C and PLD in liver macrophages labelled with [3H]arachidonic acid. Zymosan, phorbol 12-myristate 13-acetate (PMA), A23187 and fluoride but not arachidonic acid or lipopolysaccharide (LPS) induce an activation of PLD ([3H]phosphatidylethanol (PEt) accumulation). An activation of PLC ([3H]diacylglycerol (DAG) accumulation) is measured with zymosan, PMA and fluoride but not with A23187, LPS or arachidonic acid whereas inositol phosphates are formed with zymosan, only. Removal of extracellular calcium reduces the formation of [3H]PEt and [3H]DAG while pretreatment of the cells with dexamethasone reduces [3H]PEt formation, only. PMA- and zymosan-induced activation of PLD and PMA-induced activation of PLC both seem to be mediated by protein kinase (PK) C-β whereas zymosan-induced activation of PLC is negatively controlled by PKC-δ. We could furthermore present evidence that the release of [3H]arachidonic acid in these cells occurs independent of an activation of PLD.

Cotranslational chaperones assist in de novo folding of nascent polypeptides in all organisms. In yeast, the heterodimeric ribosome-associated complex (RAC) forms a unique chaperone triad with the Hsp70 homologue Ssb. We report the X-ray structure of full length Ssb in the ATP-bound open conformation at 2.6 Å resolution and identify a positively charged region in the α-helical lid domain (SBDα), which is present in all members of the Ssb-subfamily of Hsp70s. Mutational analysis demonstrates that this region is strictly required for ribosome binding. Crosslinking shows that Ssb binds close to the tunnel exit via contacts with both, ribosomal proteins and rRNA, and that specific contacts can be correlated with switching between the open (ATP-bound) and closed (ADP-bound) conformation. Taken together, our data reveal how Ssb dynamics on the ribosome allows for the efficient interaction with nascent chains upon RAC-mediated activation of ATP hydrolysis., In yeast, the heterodimeric ribosome-associated complex (RAC) acts in concert with the Hsp70 protein Ssb, forming a unique chaperone triad. Here the authors use structural and biochemical approaches to shed light on how translation and folding are coupled in eukaryotes.

Legionella is a pathogenic Gram-negative bacterium that can multiply inside of eukaryotic cells. It translocates numerous bacterial effector proteins into target cells to transform host phagocytes into a niche for replication. One effector of Legionella pneumophila is the glucosyltransferase Lgt1, which modifies serine 53 in mammalian elongation factor 1A (eEF1A), resulting in inhibition of protein synthesis and cell death. Here, we demonstrate that similar to mammalian cells, Lgt1 was severely toxic when produced in yeast and effectively inhibited in vitro protein synthesis. Saccharomyces cerevisiae strains, which were deleted of endogenous eEF1A but harbored a mutant eEF1A not glucosylated by Lgt1, were resistant toward the bacterial effector. In contrast, deletion of Hbs1, which is also an in vitro substrate of the glucosyltransferase, did not influence the toxic effects of Lgt1. Serial mutagenesis in yeast showed that Phe(54), Tyr(56) and Trp(58), located immediately downstream of serine 53 of eEF1A, are essential for the function of the elongation factor. Replacement of serine 53 by glutamic acid, mimicking phosphorylation, produced a non-functional eEF1A, which failed to support growth of S. cerevisiae. Our data indicate that Lgt1-induced lethal effect in yeast depends solely on eEF1A. The region of eEF1A encompassing serine 53 plays a critical role in functioning of the elongation factor.

Carbon monoxide (CO) is an endogenous gaseous transmitter that exerts antiproliferative effects in many cell types, but effects of CO on the translational machinery are not described. We examined the effects of the carbon monoxide releasing molecule-2 (CORM-2) on critical steps in translational signaling and global protein synthesis in pancreatic stellate cells (PSCs), the most prominent collagen-producing cells in the pancreas, whose activation is associated with pancreatic fibrosis. PSCs were isolated from rat pancreatic tissue and incubated with CORM-2. CORM-2 prevented the decrease in the phosphorylation of eukaryotic elongation factor 2 (eEF2) caused by serum. By contrast, the activation dependent phosphorylation of initiation factor 4E-binding protein 1 (4E-BP1) was inhibited by CORM-2 treatment. The phosphorylation of eukaryotic initiation factor 2α (eIF2α) and eukaryotic initiation factor 4E (eIF4E) were not affected by CORM-2 treatment. In consequence, CORM-2 mediated eEF2 phosphorylation and inactivation of 4E-BP1 suppressed global protein synthesis. These observations were associated with inhibition of phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) signaling and increased intracellular calcium and cAMP levels. The CORM-2 mediated inhibition of protein synthesis resulted in downregulation of cyclin D1 and cyclin E expression, a subsequent decline in the phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and cell growth arrest at the G(0)/G(1) phase checkpoint of the cell cycle. Our results suggest the therapeutic application of CO releasing molecules such as CORM-2 for the treatment of fibrosis, inflammation, cancer, or other pathologic states associated with excessive protein synthesis or hyperproliferation. However, prolonged exogenous application of CO might also have negative effects on cellular protein homeostasis.

Mammalian ribosome-associated complex (mRAC), consisting of the J-domain protein MPP11 and the atypical Hsp70 homolog (70-homolog) Hsp70L1, can partly complement the function of RAC, which is the homologous complex from Saccharomyces cerevisiae. RAC is the J-domain partner exclusively of the 70-homolog Ssb, which directly and independently of RAC binds to the ribosome. We here show that growth defects due to mRAC depletion in HeLa cells resemble those of yeast strains lacking RAC. Functional conservation, however, did not extend to the 70-homolog partner of mRAC. None of the major human 70-homologs was able to complement the growth defects of yeast strains lacking Ssb or was bound to ribosomes in an Ssb-like manner. Instead, our data suggest that mRAC was a specific partner of human Hsp70 but not of its close homolog Hsc70. On a mechanistic level, ATP binding, but not ATP hydrolysis, by Hsp70L1 affected mRAC's function as a J-domain partner of Hsp70. The combined data indicate that, while functionally conserved, yeast and mammalian cells have evolved distinct solutions to ensure that Hsp70-type chaperones can efficiently assist the biogenesis of newly synthesized polypeptide chains.

Treatment of macrophages with zymosan, 4 beta-phorbol 12-myristate 13-acetate (PMA) and fluoride but not with A 23187 or arachidonic acid (delta Ach) leads to a generation of diacylglycerol (acyl2Gro). Formation of inositol phosphates is achieved with zymosan, only. An elevation of intracellular calcium is obtained with zymosan and A 23187 but not with PMA, fluoride or delta Ach. Prior treatment of the cells with phorbol ester for 3 h which has been shown recently to result in a down-regulation of protein kinase (PK) C-beta but not PKC-delta [Duyster, J., Schwende, H., Fitzke, E., Hidaka H. & Dieter P. (1993) Biochem. J. 292, 203-207] has no effect on the zymosan-induced formation of acyl2Gro or inositol phosphates but inhibits the PMA-induced generation of acyl2Gro. Down-regulation of PKC-delta by prior phorbol ester treatment for 24 h augments the zymosan-induced generation of acyl2Gro and inositol phosphates. The acyl2Gro lipase inhibitor RG 80267 inhibits the PMA-induced and fluoride-induced generation of prostaglandin (PG) E2, reduces the zymosan-induced release of PGE2 by 50% but has no effect on PGE2 formation of unstimulated, A 23187-treated or delta Ach-treated cells. Furthermore, RG 80267 enhances accumulation of delta Ach-labeled acyl2Gro in response to zymosan, PMA and fluoride. These data indicate that zymosan activates a phosphatidylinositol 4,5-bisphosphate-specific phospholipase (PL) C, that generation of acyl2Gro by PMA and fluoride occurs via hydrolysis of other phospholipids, that PKC-beta is involved in the PMA-induced generation of acyl2Gro and PKC-delta negatively modulates the zymosan-induced activation of PLC and PMA and fluoride induce a liberation of delta Ach from acyl2Gro, A 23187 activates the PLA2 pathway and zymosan stimulates both, the acyl2Gro- and PLA2-pathway.

In contrast with protein kinase C (PKC)-beta, PKC-delta is exclusively detectable in the membrane fraction of liver macrophages. After long-term treatment with phorbol 12-myristate 13-acetate (PMA) PKC-beta is depleted faster (within 3 h) than PKC-delta (> 7h). Simultaneously, pretreatment with PMA for 3 h inhibits the PMA- and zymosan-induced generation of superoxide and the PMA-induced formation of prostaglandin (PG) E2, whereas a preincubation of more than 7 h is required to affect the zymosan-induced release of PGE2 and inositol phosphates. These results support an involvement of PKC-beta in the PMA-induced activation of the arachidonic acid cascade and in superoxide formation and imply an involvement of PKC-delta in zymosan-induced phosphoinositide hydrolysis and PGE2 formation. Two phorbol ester derivates, sapintoxin A (SAPA) and 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA), which have been previously reported to activate preferentially PLC-beta but not PKC-delta in vitro [Ryves, Evans, Olivier, Parker and Evans (1992) FEBS Lett. 288, 5-9], induce the formation of PGE2 and superoxide, down-regulate PKC-delta and potentiate inositol phosphate formation in parallel SAPA, but not DOPPA, down-regulates PKC-beta and inhibits the PMA-induced formation of eicosanoids and superoxide.

Over-expression of human phospholipase C-gamma 2 in murine NIH 3T3 fibroblasts has been shown to result in an increased platelet-derived growth factor-mediated formation of inositol phosphates. Here we show that phospholipase C-gamma 2 over-expression is associated with an increased platelet-derived growth factor-mediated release of arachidonic acid, prostaglandin E2, 6-keto prostaglandin F1 alpha and prostaglandin F2 alpha. The phorbol ester, calcium ionophore- and fluoride-induced release of arachidonate and its metabolites is not affected by phospholipase C-gamma 2 over-expression. Over-expression of phospholipase C-gamma 2 is also associated with an enhancement of platelet-derived growth factor-induced change in intracellular Ca2+. These results demonstrate that stimulation of recombinant human phospholipase C-gamma 2 induces a change in the intracellular Ca2+ concentration, a release of arachidonic acid and formation of prostaglandins in NIH 3T3 cells. In control cells platelet-derived growth factor-induced activation of arachidonic acid cascade is rate-limited by the endogenous phospholipase C.

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.

Yeast senses the availability of external energy sources via multiple interconnected signaling networks. One of the central components is SNF1, the homolog of mammalian AMP-activated protein kinase, which in yeast is essential for the expression of glucose-repressed genes. When glucose is available hyperphosphorylated SNF1 is rendered inactive by the type 1 protein phosphatase Glc7. Dephosphorylation requires Reg1, which physically targets Glc7 to SNF1. Here we show that the chaperone Ssb is required to keep SNF1 in the nonphosphorylated state in the presence of glucose. Using a proteome approach we found that the Δssb1Δssb2 strain displays alterations in protein expression and suffers from phenotypic characteristics reminiscent of glucose repression mutants. Microarray analysis revealed a correlation between deregulation on the protein and on the transcript level. Supporting studies uncovered that SSB1 was an effective multicopy suppressor of severe growth defects caused by the Δreg1 mutation. Suppression of Δreg1 by high levels of Ssb was coupled to a reduction of Snf1 hyperphosphorylation back to the wild-type phosphorylation level. The data are consistent with a model in which Ssb is crucial for efficient regulation within the SNF1 signaling network, thereby allowing an appropriate response to changing glucose levels.

Summary Two new potent protein kinase C inhibitors, RO 31-8220 and RO 31-7549, and staurosporine were found to inhibit dose-dependently the phorbol ester-induced formation of prostaglandin E2and superoxide in cultured liver macrophages. Prostaglandin E2formation from exogenously added arachidonate was not affected by these compounds. The zymosan-induced formation of inositol phosphates was decreased by simultaneous addition of phorbol ester and was enhanced by prior desensitization of protein kinase C indicating that protein kinase C negatively modulates phospholipase C activation in these cells. While staurosporine suppressed almost totally the zymosan-induced formation of inositol phosphates, RO 31-8220 and RO 31-7549 inhibited the protein kinase C-mediated effect on inositol phosphate formation, only. Phagocytosis of zymosan was not affected by RO 31-8220 and RO 31-7549 but was decreased by staurosporine. These results demonstrate that two new potent protein kinase C inhibitors, RO 31-8220 and RO 31-7549, are more selective in their actions as staurosporine and are useful tools to determine an involvement of protein kinase C in cellular systems.

The biosynthesis of gangliosides was studied in developing biliary cirrhosis in rats 14, 28, and 42 days after bile duct obstruction. The total content and patterns of gangliosides in livers and sera, and the activity of six hepatic ganglioside syn-thases in a cell-free system were determined. Up to 7-fold increased synthase activities were strictly correlated in time and extent with increased total contents of gangliosides in liver and serum. In addition, altered patterns of serum gangliosides were observed. The results clearly demonstrate that the liver is the main source of elevated serum gangliosides in biliary cirrhosis in the rat. Increased hepatic biosynthesis and the secretion of gangliosides into the serum appear to be an important pathogenetic event. Alterations of hepatic enzyme activities indicate that GL 2 and GM 3 synthase regulate total hepatic ganglioside content. However, certain abnormalities in ganglioside patterns which were observed in the liver and sera of cirrhotic animals can not be explained by changes in hepatic enzyme activity. They indicate additional pathobiochemical mechanisms to be involved, e.g., altered hepatocellular processing and/or impaired secretion into bile.

The two long-chain alkylamines RO 31-4493 and RO 31-4639 inhibit in a concentration-dependent manner the zymosan-induced release of arachidonic acid, the conversion of arachidonic acid into thromboxane, prostaglandin E2 and D2 and the uptake and incorporation of exogenously added arachidonate into membrane lipids of liver macrophages. The generation of superoxide and the formation of inositol phosphates is not influenced by both agents. These results suggest a rather specific interaction of RO 31-4493 and RO 31-4639 with enzymes involved in the cellular metabolism of arachidonic acid.

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.

Kupffer cells (liver macrophages) possess the ability to secrete a wide array of biologically active compounds including reactive oxygen species, nitric oxide (NO), eicosanoids and cytokines1–4. The synthesis and release of these mediators has been shown to be elicited by a number of agents including phagocytotic material (zymosan, viruses, bacteria), tumor promoting agents (phorbol ester) and other biologically active substances1–5.

In order to gain insight into the role of macrophages in human lung carcinomas, we investigated material from 35 lung carcinomas and 5 healthy lungs with 4 different antibodies (CD68, MRP8, MRP14, 27E10) recognizing different macrophage subtypes. Infiltration with CD68-positive macrophages was highest and comparable in healthy lungs and lung carcinomas. Compared to healthy lungs, the infiltration of MRP8- and MRP14-positive macrophages was reduced in lung carcinomas while the number of 27E10-positive cells was enhanced. No difference in the infiltration of macrophages was observed between the different histological subtypes of carcinomas such as squamous carcinoma, small lung carcinoma, adenocarcinoma and bronchio-alveolar carcinoma. Furthermore, we present a highly suitable technique for the isolation and enrichment of macrophages from human lung carcinomas resulting in a 5–10 fold enrichment and a yield of e.g. 2–3×10 6 27E10-positive macrophages/g tumor biopsy. Together with the recent findings that 27E10-positive macrophages are prevalent in early acute inflammation and release cytotoxic mediators [9] and to inhibit tumor cell proliferation [6] our findings suggest that 27E10-positive macrophages may play a role in antitumor cytotoxicity in human lung carcinomas.

The liberation of arachidonic acid (AA) is thought to be the rate-limiting step in prostanoid synthesis of rat-liver macrophages (RLM)1. The best known pathway for AA-liberation is elicited by the activation of a phospholipase (PL)A2 hydrolysing the ester-bond in sn-2-position of phospholipids2. It has been shown that RLM express a cPLA2 very similar to cPLA2 recently identified in U937 cells2: the enzyme has an apparant molecular weight of 100 kDa in SDS-polyacrylamide-gelelectrophoresis (PAGE), an alkaline pH-optimum, is strictly dependent on the presence of Ca2+ in the range from 10−7–10−6 M and shows a Ca2+-dependent translocation to cellular membranes1,2. cPLA2 phosphorylation by mitogen activated protein (MAP) kinase is also discussed as a mechanism for cPLA2 activation3. Another pathway leading to AA-release in RLM involves the activation of a PLC leading to free diacylglycerol (DAG), which serves as substrate for DAG-lipase to liberate free AA4.

Liver macrophages possess the ability to secrete a wide array of biologically active compounds including cytokines, nitric oxide, oxygen radicals and eicosanoids. Eicosanoid formation has been shown to be elicited by a number of particulate (bacteria, viruses, zymosan, immune complexes) and soluble (phorbol ester, PAF, C3a, fluoride, calcium ionophore, LPS, cytokines) agents. The release of arachidonic acid (AA) and eicosanoids in these cells has been shown to be controlled by glucocorticoids (1), intracellular pH (2), calcium ions (3,4), protein kinase (PK) C (5–7) and albumin in the cell media (8).

Liver macrophages possess the ability to secrete a wide array of biologically active compounds including reactive oxygen species, nitric oxide (NO), eicosanoids and cytokines1–4. The synthesis and release of these mediators has been shown to be elicited by a number of agents including phagocytotic material (zymosan, viruses, bacteria), tumor promoting agents (phorbol ester) and other biologically active substances1–5.

Rat liver macrophages (RLM), activated by a variety of agents, are known to release various eicosanoids including prostaglandin (PG)E2 and PGD2 1. The formation of prostanoids is strictly dependent on Ca2+ and is thought to be determined by the activation of a phospholipase (PL)A2 2 and the expression of prostaglandin H synthase (PGHS)-2. It has been shown that RLM express an intracellular cPLA2 which is strictly Ca2+-dependent in the range from 10-7-10-6 M and which shows a Ca2+-dependent translocation to cellular membranes3,4. cPLA2 phosphorylation has been shown to be most likely catalysed by mitogen activated protein (MAP) kinase4. Further evidence has been provided that in addition to regulation by cPLA2, release of prostanoids in RLM is also dependent on the expression of PGHS-25.

The formation and accumulation of reactive oxygen species within liver macrophages and their release into the medium were determined by lucigenin-enhanced chemiluminescence, the reduction of cytochrome c, the formation of formazan from nitroblue tetrazolium and the fluorescence of 5- (and 6)-carboxy-2',7'-dichlorodihydrofluorescein. Zymosan, phorbol ester and fluoride induced the formation and accumulation of oxygen radicals intra- and extracellularly, ionomycin and lipopolysaccharide led to an intracellular accumulation of oxygen radicals, while after arachidonic acid and tumor necrosis factor-alpha, no reactive oxygen species were formed. While zymosan and phorbol ester predominantly induced the formation and release of superoxide, hydroperoxide was the main form released by fluoride. These results indicate that agents of different biological potencies induce reactive oxygen species within and/or outside liver macrophages and that different techniques must be used to detect different oxygen species within and outside cells.

Addition of BAPTA/AM to liver macrophages lowered the level of [Ca2+]i and induced a translocation and inactivation of protein kinase C. The phorbol ester- and zymosan-induced release of arachidonic acid, prostaglandin E2 and superoxide, the formation of inositol phosphates upon addition of zymosan and the lipopolysaccharide-induced synthesis of TNF-alpha was inhibited by pretreatment of the cells with BAPTA/AM. Simultaneous addition of A23187 to elevate [Ca]i could not reverse the inhibitory effect of BAPTA. Phagocytosis of zymosan and formation of prostaglandin E2 from exogenously added arachidonic acid or upon addition of A 2187 was not altered by BAPTA/AM. No protein kinase C activity could be measured in homogenates obtained from BAPTA/AM-pretreated cells. These results indicate that the action of BAPTA in eucaryotic cells is not limited to its chelating effect on calcium but that BAPTA leads to a translocation and inactivation of protein kinase C.

Total content, pattern and transport by lipoproteins of gangliosides have been studied in the sera of 10 patients with hypercholesterolemia and manifest cardiovascular disease. Half of the patients with hypercholesterolemia and 3 healthy controls were treated with heparin-induced extracorporeal LDL precipitation (HELP). In the sera of the untreated group total gangliosides and cholesterol were elevated about 2-fold. Ratios of normal ganglioside components were not altered and abnormal ganglioside species not detected. Treatment with HELP resulted in an almost selective removal of lipid-bound sialic acid carried on LDL. The re-increase of total serum gangliosides was strictly correlated to that of LDL-cholesterol and apolipoprotein B. Total gangliosides and ratios of individual components carried on single LDL- and HDL-particles were not altered by the HELP treatment. Our results indicate that gangliosides are excreted into the serum along with nascent apolipoprotein B-containing lipoproteins, which are of hepatic origin. In hypercholesterolemia excretion of gangliosides into the circulation is elevated and surplus of circulating gangliosides is bound to increased numbers of ‘atherogenic’ LDL. Biosynthesis of different ganglioside components, most probably by the liver, and total amount of gangliosides bound to lipoprotein particles seem not to be altered.

De novo synthesis and excretion into perfusate and bile fluid of hepatic gangliosides were studied in isolated perfused rat livers. Addition of N-acetyl-[6-3H(n)]D-mannosamine to the perfusate resulted in radioactive synthesis of at least eight gangliosides labeled in their sialic acid residues. About 10% of total de novo synthesized gangliosides were excreted into the perfusate, less than 1% into the bile fluid. Labeled gangliosides were tentatively identified by cochromatography with known standards. All of them are known to occur in rat liver and sera. The results indicate that most, if not all, normal serum gangliosides are synthesized in the liver; excretion with bile fluid is negligible. They explain previous observations, and indicate clinical implications, which are discussed.

Glucocorticoids inhibited the zymosan-induced formation of inositol phosphates in macrophages. No inhibition was observed with progesterone. Inhibitors of protein (cycloheximide) and RNA (actinomycin D) synthesis exhibited similar inhibitory effects. The activity of phospholipase C in subcellular fractions was not altered by hormone treatment of the cells. However, the incorporation of inositol into membrane lipids was reduced by dexamethasone. These data indicate that glucocorticoids are able to inhibit the formation of inositol phosphates; the effect of the hormone is rather due to an inhibition of the incorporation of inositol in membrane lipids than to an inhibition of phospholipase C. The anti-inflammatory action of glucocorticoids may, therefore, also be attributed to their effect on the polyphosphoinositide cycle and inositol phosphate-mediated processes.

Concentrations, patterns and distribution in different lipoprotein classes of human serum gangliosides were investigated in acute and chronic liver diseases of different etiologies. The total concentrations of gangliosides were moderately elevated in sera of patients with cirrhosis and acute B or NANB virus hepatitis, but almost 3-fold in those with severe cholestasis. Up to three unknown gangliosides appeared in the sera of six out of nine patients with alcoholic cirrhosis. They accounted for 11-27% of total serum gangliosides. In acute viral hepatitis very small amounts of these gangliosides were inconsistently detected. In severe cholestasis (bilirubin greater than 10 mg/dl) the distribution of serum gangliosides was altered in different lipoprotein classes including lipoprotein X (LP(x)). The results indicate that the liver produces serum gangliosides. The diseased liver is supposed to affect the total concentration, pattern and distribution of serum gangliosides in different lipoprotein classes as a result of at least two different pathogenetic events: the qualitative and quantitative alterations of their biosynthesis and secretion into the circulation (cirrhosis); and the alteration of lipoprotein metabolism following cholestasis.

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 uniformly 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.

The total content and pattern of gangliosides were determined in the unfractionated sera of 11 healthy human adults and in isolated lipoproteins. The total content of lipid-bound sialic acid was 10.5 +/- 3.2 nmol/ml serum. The ganglioside profile consisted of more than ten different components. The major ganglioside was GM3, followed by GD3, GD1a, GM2, GT1b, MG-3 (sialosyllactoneotetraosylceramide), GD1b and GQ1b. Traces of four additional gangliosides could not be quantified reliably. Ganglioside patterns did not vary in sera taken from healthy adults of different age and sex. Approximately 98% of human serum gangliosides were transported by serum lipoproteins, predominantly by LDL (66%), followed by HDL (25%) and VLDL (7%). The quantitative distribution of individual gangliosides in VLDL and LDL was almost the same as that in the unfractionated serum; some differences existed with the ganglioside profile in HDL.

Over-expression of human protein kinase C-alpha in murine NIH 3T3 fibroblasts is associated with an increased platelet-derived growth factor- and phorbol ester-mediated formation of prostaglandins, whereas the calcium ionophore-induced release of arachidonic acid metabolites is unaffected; however, the differences of arachidonic acid and prostaglandin formation are much more pronounced with platelet-derived growth factor than with phorbol ester. Platelet-derived growth factor induces an identical elevation of intracellular free calcium in control and protein kinase C-alpha over-expressing cells: the phorbol ester has no effect on intracellular free calcium in both cell lines. These results demonstrate that protein kinase C-alpha may couple to arachidonic acid cascade in NIH 3T3 fibroblasts.

Clostridium pasteurianum is able to build up about 100-fold gradients of methylammonium across the cell membrane. Methylammonium enters the cell by means of a carrier as shown by the energy requirement, saturation kinetics and a pH profile with a narrow maximum between pH 6.2 and 6.8. The methylammonium transport (apparent K m = 150 μ M, V = 100 μ mol/min per g dry weight) is competitively inhibited by ammonium (apparent K i = 9 μ M). The low K i value and the observation that methylammonium cannot serve as a carbon or nitrogen source for Cl. pasteurianum strongly indicate that ammonium rather than methylammonium is the natural substrate. Uncouplers and inhibitors of energy metabolism or of the membrane-bound ATPase inhibit transport. Cl. pasteurianum maintains a membrane potential (interior negative) in the range 80–130 mV. This membrane potential was identified as the energy source: the same agents that block transport also decrease the membrane potential, and artificial generation of a membrane potential (by addition of valinomycin to K + -loaded cells) induces concentrative uptake of methylammonium. Thus NH + 4 (or CH 3 NH 3 + ) must be the transported species. Digestion of the cell wall by lysozyme does not abolish the transport activity.

Clostridiumpasteurianum is able to take up NH4+ and CH3NH3+ against concentration gradients. Uptake of CH3NH3+ is abolished by NH4+ and partially inhibited by dinitrophenol. C.pasteurianum membranes are permeabilized for NH4+ by valinomycin. These results are regarded as evidence for an ammonium translocase in membranes otherwise only slightly permeable for NH3.

In this study we have verified the existence of a cytosolic phospholipase A2 (cPLA2) in rat-liver macrophages. Stimulation of these cells with phorbol 12-myristate 13-acetate (PMA), zymosan and lipopolysaccharide (LPS), but not with the Ca(2+)-ionophore A23187, leads to phosphorylation of cPLA2 and activation of mitogen-activated protein (MAP) kinase, supporting the hypothesis that MAP kinase is involved in cPLA2 phosphorylation. We show furthermore, that the tyrosine kinase inhibitor genistein prevents the LPS- but not the PMA- or zymosan-induced phosphorylation of cPLA2 and activation of MAP kinase, indicating that tyrosine kinases participate in LPS- but not in PMA- and zymosan-induced cPLA2 phosphorylation and MAP kinase activation. Phosphorylation of cPLA2 does not strongly correlate with stimulation of the arachidonic acid (AA) cascade: (1) A23187, a potent stimulator of AA release, fails to induce cPLA2 phosphorylation; (2) withdrawal of extracellular Ca2+, which inhibits PMA-stimulated AA release (Dieter, Schulze-Specking and Decker (1988) Eur. J. Biochem. 177, 61-67), has no effect on PMA-induced phosphorylation of cPLA2; (3) LPS induces cPLA2 phosphorylation within minutes, whereas increased AA release upon treatment with LPS is detectable for the first time after 4 h; and (4) genistein, which prevents LPS-induced cPLA2 phosphorylation, does not inhibit AA release in response to LPS. From these data we suggest that a rise in intracellular Ca2+, but not phosphorylation of cPLA2, is essential for activation of the AA cascade in rat-liver macrophages.