Thymic stromal lymphopoietin deficiency attenuates experimental autoimmune encephalomyelitis.

In the present study we examined the role of thymic stromal lymphopoietin (TSLP) in experimental autoimmune encephalomyelitis (EAE). Here, we report that TSLP knock-out (KO) mice display a delayed onset of disease and an attenuated form of EAE. This delayed onset was accompanied by a reduced number of encephalitogenic T helper type 1 (Th1) cells in the central nervous system (CNS) of TSLP KO mice. In addition, CD4(+) and CD8(+) T cells from CNS of TSLP KO mice show a reduced activation status in comparison to wild-type mice. It is noteworthy that we could also show that lymph node cells from TSLP KO mice expanded less efficiently and that interleukin (IL)-6-, interferon (IFN)-γ and tumour necrosis factor (TNF)-α levels were reduced. Furthermore, CD3(+) T cells isolated in the preclinical phase from myelin oligodendrocyte glycoprotein peptide 35-55 (MOG(35-55))-immunized TSLP KO mice showed a reduced response after secondary exposure to MOG(35-55), indicating that differentiation of naive T cells into MOG(35-55)-specific effector and memory T cells was impaired in KO mice. The addition of recombinant TSLP enhanced T cell proliferation during MOG(35-55) restimulation, showing that T cells also respond directly to TSLP. In summary, these data demonstrate that expression of, and immune activation by, TSLP contributes significantly to the immunopathology of EAE.

Soluble CD83 ameliorates experimental colitis in mice.

The physiological balance between pro- and anti-inflammatory processes is dysregulated in inflammatory bowel diseases (IBD) as in Crohn's disease and ulcerative colitis. Conventional therapy uses anti-inflammatory and immunosuppressive corticosteroids to treat acute-phase symptoms. However, low remission rate and strong side effects of these therapies are not satisfying. Thus, there is a high medical need for new therapeutic strategies. Soluble CD83, the extracellular domain of the transmembrane CD83 molecule, has been reported to have interesting therapeutic and immunosuppressive properties by suppressing dendritic cell (DC)-mediated T-cell activation and inducing tolerogenic DCs. However, the expression and function of CD83 in IBD is still unknown. Here, we show that CD83 expression is upregulated by different leukocyte populations in a chemical-induced murine colitis model. Furthermore, in this study the potential of sCD83 to modulate colitis using an experimental murine colitis model was investigated. Strikingly, sCD83 ameliorated the clinical disease symptoms, drastically reduced mortality, and strongly decreased inflammatory cytokine expression in mesenteric lymph nodes and colon. The infiltration of macrophages and granulocytes into colonic tissues was vigorously inhibited. Mechanistically, we could show that sCD83-induced expression of indolamine 2,3-dioxygenase is essential for its protective effects.

Altered membrane fluidity and lipid raft composition in presenilin-deficient cells.

The pathology of Alzheimer's disease is closely connected with lipid metabolism. Processing of amyloid precursor protein (APP) is sensitive to membrane alterations in levels of cholesterol and gangliosides. As cholesterol and gangliosides are major components of rafts and BACE I and gamma-secretase are supposed to be localized to rafts there might be a yet unknown biological function underlying this connection. Increasing evidence shows a close connection between cholesterol homeostasis and APP processing and Abeta production respectively. We measured membrane fluidity by anisotropy determination, isolated detergent resistant membrane (DRM) fractions from membrane preparations and determined cholesterol content of these fractions by a coupled enzymatic assay. We found membrane fluidity to be changed in mouse embryonic fibroblasts (MEF) PS1/2 -/- along with altered cholesterol content in DRM fraction of these cells. In addition, total ganglioside levels were enhanced in absence of presenilin (PS).

Effects of KHEYLRFamide and KNEFIRFamide on cyclic adenosine monophosphate levels in Ascaris suum somatic muscle.

KHEYLRF-NH(2) (AF2) is a FMRFamide-related peptide (FaRP) present in parasitic and free-living nematodes. At concentrations as low as 10 pM, AF2 induces a biphasic tension response, consisting of a transient relaxation followed by profound excitation, in neuromuscular strips prepared from Ascaris suum. In the present study, the effects of AF2 on cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and inositol-1,4,5-triphosphate (IP(3)) levels were measured following muscle tension recordings from 2 cm neuromuscular strips prepared from adult A. suum. AF2 induced a concentration- and time-dependent increase in cAMP, beginning at 1 nM; cAMP levels increased by 84-fold following 1 h exposure to 1 microM AF2. cGMP and IP(3) levels were unaffected by AF2 at concentrations

Prolonged stationary-phase incubation selects for lrp mutations in Escherichia coli K-12.

Evolution by natural selection occurs in cultures of Escherichia coli maintained under carbon starvation stress. Mutants of increased fitness express a growth advantage in stationary phase (GASP) phenotype, enabling them to grow and displace the parent as the majority population. The first GASP mutation was identified as a loss-of-function allele of rpoS, encoding the stationary-phase global regulator, sigma(S) (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757-1760, 1993). We now report that a second global regulator, Lrp, can also play a role in stationary-phase competition. We found that a mutant that took over an aged culture of an rpoS strain had acquired a GASP mutation in lrp. This GASP allele, lrp-1141, encodes a mutant protein lacking the critical glycine in the turn of the helix-turn-helix DNA-binding domain. The lrp-1141 allele behaves as a null mutation when in single copy and is dominant negative when overexpressed. Hence, the mutant protein appears to retain stability and the ability to dimerize but lacks DNA-binding activity. We also demonstrated that a lrp null allele generated by a transposon insertion has a fitness gain identical to that of the lrp-1141 allele, verifying that cells lacking Lrp activity have a competitive advantage during prolonged starvation. Finally, we tested by genetic analysis the hypothesis that the lrp-1141 GASP mutation confers a fitness gain by enhancing amino acid catabolism during carbon starvation. We found that while amino acid catabolism may play a role, it is not necessary for the lrp GASP phenotype, and hence the lrp GASP phenotype is due to more global physiological changes.

Mutations enhancing amino acid catabolism confer a growth advantage in stationary phase.

Starved cultures of Escherichia coli undergo successive rounds of population takeovers by mutants of increasing fitness. These mutants express the growth advantage in stationary phase (GASP) phenotype. Previous work identified the rpoS819 allele as a GASP mutation allowing cells to take over stationary-phase cultures after growth in rich media (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757-1760, 1993). Here we have identified three new GASP loci from an aged rpoS819 strain: sgaA, sgaB, and sgaC. Each locus is capable of conferring GASP on the rpoS819 parent, and they can provide successively higher fitnesses for the bacteria in the starved cultures. All four GASP mutations isolated thus far allow for faster growth on both individual and mixtures of amino acids. Each mutation confers a growth advantage on a different subset of amino acids, and these mutations act in concert to increase the overall catabolic capacity of the cell. We present a model whereby this enhanced ability to catabolize amino acids is responsible for the fitness gain during carbon starvation, as it may allow GASP mutants to outcompete the parental cells when growing on the amino acids released by dying cells.

Synthesis and biological activity of anthelmintic thiadiazoles using an AF-2 receptor binding assay.

Following our discovery of the strong binding of thiadiazole 1 to the AF-2 neuropeptide receptor of gastrointestinal nematodes (e.g., Ascaris suum), we prepared two series of analogs. Only the series containing the thiadiazole ring had potencies comparable to that of compound 1. Analog 50 exhibited an apparent potency in the AF-2 binding assay 300 times that of compound 1.

Dual localization of squalene epoxidase, Erg1p, in yeast reflects a relationship between the endoplasmic reticulum and lipid particles.

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 x g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Delta24-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.

Export of steryl esters from lipid particles and release of free sterols in the yeast, Saccharomyces cerevisiae.

Fatty acyl esters of the yeast specific sterol, ergosterol, are exclusively stored in lipid particles. Under conditions of sterol deficiency, e.g., in the presence of terbinafine, an inhibitor of fungal squalene epoxidase, steryl esters are hydrolyzed, and sterols are set free for membrane formation. Lipid particles do not contain steryl-ester hydrolase activity themselves; the highest specific activity of this enzyme is found in the plasma membrane. Therefore, steryl esters have to be exported from lipid particles to their site of hydrolytic cleavage. This process of translocation and metabolic conversion was studied in vivo. Addition of nocodazole to terbinafine-treated cells did not disturb the mobilization of steryl esters, indicating that this process is not mediated by microtubuli-dependent vesicle flux. Under the influence of inhibitors of cellular energy production (azide and fluoride) and protein biosynthesis (cycloheximide) mobilization of steryl esters came to an halt. These results support the view that ongoing membrane proliferation may be a driving force for the release of sterols from steryl esters of lipid particles.

Characterization of lipid particles of the yeast, Saccharomyces cerevisiae.

Lipid particles of the yeast, Saccharomyces cerevisiae, were isolated to high purity and their components were analysed. The hydrophobic core of this organelle consists of triacylglycerols and steryl esters, which are almost exclusively located to that compartment. Lipid particles are stabilized by a surface membrane consisting of phospholipids and proteins. Electron microscopy confirmed the purity of the preparations and the proposed structure deduced from biochemical experiments. Major proteins of lipid particles have molecular weights of 72, 52, 43 and 34 kDa, respectively. The 43 kDa protein reacts with an antiserum against human apolipoprotein AII. In lipid particles of the yeast mutant strain S. cerevisiae erg6, which is deficient in sterol delta 24-methyltransferase, this protein is missing thereby identifying the protein and confirming our previous finding (Zinser et al., 1993) that sterol delta 24-methylation is associated with lipid particles. A possible involvement of surface proteins of lipid particles in the interaction with other organelles is discussed with respect to sterol translocation in yeast.

Characterization, quantification and subcellular localization of inositol-containing sphingolipids of the yeast, Saccharomyces cerevisiae.

In yeast, as in higher eukaryotic cells, sphingolipids are essential membrane components. The yeast, Saccharomyces cerevisiae, contains three classes of sphingolipids, inositolphosphorylceramide (InsPCer), mannosylinositolphosphorylceramide (ManInsPCer) and mannosyldiinositolphosphorylceramide (ManPIns2PCer). As a prerequisite to localize these sphingolipids in subcellular membranes, authentic standards of the respective lipids were isolaed and characterized using biochemical methods and electrospray ionization mass spectrometry. The complete set of yeast subcellular membranes was isolated at high purity, and sphingolipids were extracted. InsPCer, ManInsPCer, and ManPIns2PCer were separated by thin-layer chromatography, stained and densitometrically scanned along with the respective standards. These methods enable a complete overview of the subcellular distribution of yeast sphingolipids to be obtained, as far as is known, for the first time. InsPCer was highly enriched in Golgi and vacuolar membranes, whereas the largest amounts of ManInsPCer and ManPIns2PCer were found in the plasma membrane. The presence of inositol-containing sphingolipids in organelles of the protein-secretory pathway strongly supports the notion that protein secretion and intracellular trafficking of sphingolipids are linked processes.

Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH.

Escherichia coli K-12 strains and Shigella flexneri grown to stationary phase can survive several hours at pH 2 to 3, which is considerably lower than the acid limit for growth (about pH 4.5). A 1.3-kb fragment cloned from S. flexneri conferred acid resistance on acid-sensitive E. coli HB101; sequence data identified the fragment as a homolog of rpoS, the growth phase-dependent sigma factor sigma 38. The clone also conferred acid resistance on S. flexneri rpoS::Tn10 but not on Salmonella typhimurium. E. coli and S. flexneri strains containing wild-type rpoS maintained greater internal pH in the face of a low external pH than strains lacking functional rpoS, but the ability to survive at low pH did not require maintenance of a high transmembrane pH difference. Aerobic stationary-phase cultures of E. coli MC4100 and S. flexneri 3136, grown initially at an external pH range of 5 to 8, were 100% acid resistant (surviving 2 h at pH 2.5). Aerobic log-phase cultures grown at pH 5.0 were acid resistant; survival decreased 10- to 100-fold as the pH of growth was increased to pH 8.0. Extended growth in log phase also decreased acid resistance substantially. Strains containing rpoS::Tn10 showed partial acid resistance when grown at pH 5 to stationary phase; log-phase cultures showed < 0.01% acid resistance. When grown anaerobically at low pH, however, the rpoS::Tn10 strains were acid resistant. E. coli MC4100 also showed resistance at alkaline pH outside the growth range (base resistance). Significant base resistance was observed up to pH 10.2. Base resistance was diminished by rpoS::Tn10 and by the presence of Na+. Base resistance was increased by an order of magnitude for stationary-phase cultures grown in moderate base (pH 8) compared with those grown in moderate acid (pH 5). Anaerobic growth partly restored base resistance in cultures grown at pH 5 but not in those grown at pH 8. Thus, both acid resistance and base resistance show dependence on growth pH and are regulated by rpoS under certain conditions. For acid resistance, and in part for base resistance, the rpoS requirement can be overcome by anaerobic growth in moderate acid.

Two yeast peroxisomal proteins crossreact with an antiserum against human sterol carrier protein 2 (SCP-2).

An antibody raised against human sterol carrier protein 2 (SCP-2) crossreacts with two yeast peroxisomal proteins. These proteins have apparent molecular weights of 35 and 58 kDa. Subfractionation of peroxisomes revealed that the 58 kDa species is a soluble matrix protein, whereas the 35 kDa protein is membrane bound. Treatment of isolated peroxisomal membranes with 0.25 M KCl released the 35 kDa crossreactive protein into the soluble supernatant. However, lipid transfer activity could be attributed neither to the 35 kDa nor to the 58 kDa protein.

Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism.

Organelles of the yeast Saccharomyces cerevisiae were isolated and analyzed for sterol composition and the activity of three enzymes involved in sterol metabolism. The plasma membrane and secretory vesicles, the fractions with the highest sterol contents, contain ergosterol as the major sterol. In other subcellular membranes, which exhibit lower sterol contents, intermediates of the sterol biosynthetic pathway were found at higher percentages. Lipid particles contain, in addition to ergosterol, large amounts of zymosterol, fecosterol, and episterol. These sterols are present esterified with long-chain fatty acids in this subcellular compartment, which also harbors practically all of the triacylglycerols present in the cell but very little phospholipids and proteins. Sterol delta 24-methyltransferase, an enzyme that catalyzes one of the late steps in sterol biosynthesis, was localized almost exclusively in lipid particles. Steryl ester formation is a microsomal process, whereas steryl ester hydrolysis occurs in the plasma membrane and in secretory vesicles. The fact that synthesis, storage, and hydrolysis of steryl esters occur in different subcellular compartments gives rise to the view that ergosteryl esters of lipid particles might serve as intermediates for the supply of ergosterol from internal membranes to the plasma membrane.

Influence of transfected SV40 early region on growth and differentiation of human endothelial cells.

Endothelial cells isolated from human umbilical veins show a limited in vitro life span of about 40 population doublings. Cell division is dependent on the presence of endothelial cell growth factor in the culture medium. We have transfected primary endothelial cells with a plasmid containing the early region of SV40 virus. Large T positive cells were obtained which grew in the absence of endothelial cell growth factor at low serum concentrations and showed a prolonged lifespan. Expression of von Willebrand factor and SV40 large T antigen was detected simultaneously in transfected cells.

Mitochondrial membrane contact sites of yeast. Characterization of lipid components and possible involvement in intramitochondrial translocation of phospholipids.

Submitochondrial membrane fractions from yeast that are enriched in inner and outer membrane contact sites were analyzed with respect to their lipid composition. Characteristic features were the significantly reduced content of phosphatidylinositol, the decreased amount of phosphatidylcholine, and the enrichment in phosphatidylethanolamine and cardiolipin. Coisolation of phosphatidylserine synthase with the outer membrane portion and enrichment of phosphatidylserine decarboxylase in the inner membrane portion of isolated contact sites provided the basis for a metabolic assay to study phosphatidylserine transfer from the outer to the inner mitochondrial membrane via contact sites. The efficient conversion to [3H]phosphatidylethanolamine of [3H]phosphatidylserine synthesized from [3H]serine in situ supports the notion that mitochondrial membrane contact sites are zones of intramitochondrial translocation of phosphatidylserine.

Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae.

Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.

Utilization of exogenous glycerophosphodiesters and glycerol 3-phosphate by inositol-starved yeast, Saccharomyces uvarum.

Inositol-starved Saccharomyces uvarum cells hydrolyse exogenous glycerophosphodiesters to glycerol 3-phosphate and the corresponding alcohol. Glycerophosphodiesterase activity is highest with glycerophosphoinositol as the substrate, followed by glycerophosphoethanolamine and glycerophosphocholine; the artificial substrate for phosphodiesterases, bis-p-nitrophenylphosphate,is hydrolysed at a similar rate as compared with glycerophosphoinositol. Competition experiments suggest that distinct phosphodiesterases are involved in the hydrolysis of the respective substrates. An Mg2+-dependent glycerophosphate phosphohydrolase with a pH-optimum around neutral cleaves glycerol 3-phosphate to glycerol and orthophosphate. The latter is taken up into cells without first entering the pool of orthophosphate present in the growth medium. Accessibility to substrates with whole cells, adhesion of enzymes to spheroplasts, and solubilization of enzymes by treatment of whole cells with Triton X-100 under mild conditions suggest that phosphodiesterases and glycerol-3-phosphate phosphohydrolase are loosely associated with the outer side of the yeast plasma membrane. Enzyme activities are only marginal in inositol-supplemented cells, but are derepressed not only by inositol deficiency, but also by starvation of orthophosphate.

The effect of myo-inositol deficiency on phosphatases of yeast.

Activities of several phosphohydrolases are significantly enhanced when cells of the inositol-requiring yeast, Saccharomyces uvarum ATCC 9080, are deprived of inositol. This effect is most pronounced for the external acid phosphatase and cannot be explained simply by limitation of cellular growth, because starvation for vitamins or sulphate has no effect on acid phosphatase activities. Excessive secretion of acid phosphatase by spheroplasts prepared from inositol-deficient cells is greatly reduced when the spheroplast medium is supplemented with inositol and is immediately suppressed by the addition of cycloheximide. These results together with data obtained from experiments with whole cells, employing cycloheximide and actinomycin D, point to a regulatory effect of inositol limitation at the level of transcription. The external enzymes beta-D-fructofuranosidase, alpha-D-galactosidase and L-asparaginase, and the vacuolar enzyme carboxypeptidase Y are not affected by inositol deficiency indicating that inositol deficiency has no general effect on protein secretion.