Relationship of other cytoplasmic ribonucleoprotein bodies (cRNPB) to GW/P bodies.

GW/P body components are involved in the post-transcriptional -processing of messenger RNA (mRNA) through the RNA interference and 5' → 3' mRNA degradation pathways, as well as functioning in mRNA transport and stabilization. It is currently thought that the relevant mRNA silencing and degrading factors are partitioned to these cytoplasmic microdomains thus effecting post-transcriptional regulation and the prevention of accidental degradation of functional mRNA. Although much attention has focused on GW/P bodies, a variety of other cytoplasmic RNP bodies (cRNPB) also have highly specialized functions and have been shown to interact or co-localize with components of GW/P bodies. These cRNPB include neuronal transport RNP granules, stress granules, RNP-rich cytoplasmic germline granules or chromatoid bodies, sponge bodies, cytoplasmic prion protein-induced RNP granules, U bodies and TAM bodies. Of clinical relevance, autoantibodies directed against protein and miRNA components of GW/P bodies have been associated with autoimmune diseases, neurological diseases and cancer. Understanding the molecular function of GW/P bodies and their interactions with other cRNPB may provide clues to the etiology or pathogenesis of diseases associated with autoantibodies directed to these structures. This chapter will focus on the similarities and differences of the various cRNPB as an approach to understanding their functional relationships to GW/P bodies.

The role of microparticles in inflammation and thrombosis.

Microparticles (MP) are small membrane-bound vesicles that circulate in the peripheral blood and play active roles in thrombosis, inflammation and vascular reactivity. While MP can be released from nearly every cell type, most investigation has focused on MP of platelet, leucocyte and endothelial cell origin. Cells can release MP during activation or death. Flow cytometry is the usual method to quantify MP; the small size of these structures and lack of standardization in methodology complicate measurement. As MP contain surface and cytoplasmic contents of the parent cells and bear phosphatidylserine, antibodies to specific cell surface markers and annexin V can be used for identification. Through various mechanisms, MP participate in haemostasis and have procoagulant potential in disease. MP contribute to inflammation via their influence on cell-cell interactions and cytokine release, and MP also function in mediating vascular tone. In several disease states characterized by inflammation and vascular dysfunction, MP subpopulations are elevated, correlate with clinical events, and may have important roles in pathogenesis. In the rheumatic conditions such as rheumatoid arthritis and systemic lupus erythematosus, MP are potentially important markers of disease activity and have an increasingly recognized role in immunopathogenesis. It is clear that MP play an important role in atherosclerosis, and study of these structures may provide insight into the link between chronic inflammatory conditions and accelerated atherosclerosis. As biomarkers, MP allow access to usually inaccessible tissues such as the endothelium. Further research will hopefully lead to interventions targeting MP release and function.

Monocyte/macrophage-derived microparticles up-regulate inflammatory mediator synthesis by human airway epithelial cells.

Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.

The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms.

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.

Effects of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) on the expression of inflammatory cytokines and apoptosis induction in rheumatoid synovial fibroblasts and monocytes.

This study was performed to investigate whether peroxisome proliterator-activated receptor-gamma (PPAR-gamma) exerted an anti-inflammatory effect on rheumatoid synovial cells and inhibited dysregulated proliferation. The expression of PPAR-gamma mRNA in cultured human synoviocytes and THP-1 cells was analysed by RT-PCR. PPAR-gamma was expressed in normal, osteoarthritis (OA), rheumatoid arthritis (RA) synovial cells as well as a human monocytic cell line, THP-1. In RA and OA synoviocytes, the induction of inflammatory cytokine mRNA expression such as TNF-alpha and IL-1beta was significantly inhibited by the natural PPAR-gamma agonist, 15 deoxy-Delta(12,14)prostaglandin J(2)(15d-PGJ(2)). The effect of PPAR-gamma on the nuclear factor (NF)-kappaB activity was tested by electrophoretic mobility shift assay (EMSA). Both troglitazone and 15d-PGJ(2)markedly inhibited TNF-alpha-induced NF-kappaB activation at 30 microM. However, PPAR-gamma agonist neither reduced proliferation nor induced apoptosis in RA synoviocytes when measured by XTT assay and fluorescence activated cell sorter (FACS) analysis. In contrast, it induced apoptosis in a dose-dependent manner in THP-1 cells and augmented TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis as well. In conclusion, these data demonstrate that PPAR-gamma is expressed in human synoviocytes and THP-1 cells, and the PPAR-gamma activation inhibits expression of inflammatory cytokines in RA synoviocytes. Furthermore, PPAR-gamma activation induces apoptosis by itself and augments TRAIL/Apo2L-induced apoptosis in THP-1 cells. These results suggest that PPAR-gamma agonists may provide a new therapeutic approach for RA.

The nuclear receptor PPAR gamma and immunoregulation: PPAR gamma mediates inhibition of helper T cell responses.

The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily. Until recently, the genes regulated by PPARs were those believed to be predominantly associated with lipid metabolism. Recently, an immunomodulatory role for PPAR gamma has been described in cells critical to the innate immune system, the monocyte/macrophage. In addition, evidence for an antiinflammatory role of the PPAR gamma ligand, 15-deoxy-Delta 12,14-PGJ2 (15d-PGJ2) has been found. In the present studies, we demonstrate, for the first time, that murine helper T cell clones and freshly isolated splenocytes express PPAR gamma 1. The PPAR gamma expressed is of functional significance in that two ligands for PPAR gamma, 15d-PGJ2 and a thiazolidinedione, ciglitazone, mediate significant inhibition of proliferative responses of both the T cell clones and the freshly isolated splenocytes. This inhibition is mediated directly at the level of the T cell and not at the level of the macrophage/APC. Finally, we demonstrate that the two ligands for PPAR gamma mediate inhibition of IL-2 secretion by the T cell clones while not inhibiting IL-2-induced proliferation of such clones. The demonstration of the expression and function of PPAR gamma in T cells reveals a new level of immunoregulatory control for PPARs and significantly increases the role and importance of PPAR gamma in immunoregulation.

Peroxisome subpopulations of the rat liver. Isolation by immune free flow electrophoresis.

Peroxisomes (POs) are a heterogenous population of cell organelles which, in mammals, are most abundant in liver and kidney. Although they are usually isolated by differential and density gradient centrifugation, isolation is hampered by their high fragility, sensitivity to mechanical stress, and their sedimentation characteristics, which are close to those of other major organelles, particularly microsomes. Consequently, until now only the so-called "heavy" POs with a buoyant density of 1.22-1.24 g/cm(3) have been highly purified from rat liver, whereas the other subpopulations also present in that tissue have escaped adequate characterization. The purification of these subpopulations has become an essential task in view of the functional significance of POs in humans, and the putative importance of peroxisomal subpopulations in the biogenesis of this organelle. Here we used an alternative novel approach to density gradient centrifugation, called immune free flow electrophoresis (IFFE). IFFE combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It makes use of the fact that the electrophoretic mobility of a subcellular particle complexed to an antibody against the cytoplasmic domain of one of its integral membrane proteins is greatly diminished, provided that the pH of the electrophoresis buffer is adjusted to pH approximately 8.0, the pI of IgG molecules. Because of this reduced electrophoretic mobility, IgG-coupled particles can be separated in an electric field from those that are noncoupled and hence more mobile. The IFFE technique has been recently applied for isolation of regular POs (rho = 1.22-1.24 g/cm(3)) from a light mitochondrial fraction of rat liver. We succeeded in isolating different subpopulations of POs by applying IFFE to heavy, light, and postmitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The PO subfractions obtained differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic POs.

Isolation of peroxisome subpopulations from rat liver by means of immune free-flow electrophoresis.

Immune free-flow electrophoresis (IFFE) has been applied to the separation of peroxisomes (PO). IFFE is a modification of antigen-specific electrophoretic cell separation (ASECS), and combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It differs from the latter in the pH of the electrophoresis buffer, which was shifted from the physiological range (ASECS) to the pI of IgG molecules (pH approximately 8.0), thus further decreasing the mobility produced by the binding of a specific antibody. This enhances the mobility differences between IgG-coupled particles and those nondecorated, with resultant improved separation. We have now succeeded in isolating different subpopulations of PO by applying IFFE to heavy, light, and post-mitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The obtained PO subfractions differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic PO.

An 80-kDa syncytiotrophoblast alloantigen bound to maternal alloantibody in term placenta.

PROBLEM: We have shown that most of the IgG present on term syncytiotrophoblast, membrane, microvesicles is bound to an 80 kDa protein antigen (R80K). METHODS: Microvesicles were prepared from term human placenta, and the IgG eluted at pH3. RESULTS: When IgG antibody was eluted at pH3 and reacted with acid-treated vesicles of other placentae, the alloantibody always bound to the preparation from which it was obtained, but only to about 10% of acid-treated preparations from other placentae. A similar polymorphic protein found in association with IgG antibody was found in term horse placentae. Cross-reactivity of the antibodies between species was not found. Using binding of labelled antibody, complement dependent cytotoxicity and FACS two-color analysis, the human polymorphic antigen was present on peripheral blood monocytes and B-lymphocytes. The R80k antigen on intact microvesicles was resistant to trypsin, but after acid elution of IgG, trypsin released a soluble 50 kDa fragment which reacted with the acid-eluted IgG antibody. CONCLUSION: The presence of antibodies to R80K in all term placentae studied, including first pregnancies, suggests that development of this alloantibody may be a normal requirement for successful pregnancy.

The importance of tissue fixation for light microscopic immunohistochemical localization of peroxisomal proteins: the superiority of Carnoy's fixative over Baker's formalin and Bouin's solution.

We have compared the effects of fixation with three commonly used fixatives upon preservation of the antigenicity of six peroxisomal proteins in rat liver using both immunohistochemical staining and Western blotting of fixed tissue extracts. The immunoreactivity of all six peroxisomal proteins was well preserved and peroxisomes were clearly identified in material fixed in Carnoy's fixative. Moreover, the corresponding proteins stained well in Western blots prepared from extracts of Carnoy-fixed material. The intensity of the immunohistochemical staining was reduced at different rates for individual peroxisomal proteins after fixation in Baker's formalin, but peroxisomes were still well visualized with antibodies to catalase and some beta-oxidation enzymes. No evidence of immunohistochemical staining for any peroxisomal antigens was obtained after fixation in Bouin's fluid. For detection of the antibody binding sites in Carnoy's fixed material, the avidin-biotin-peroxidase complex (ABC) with aminoethyl carbazole as chromogen was found to be superior to the methods of peroxidase-antiperoxidase/diaminobenzidine and protein A-gold with silver intensification. Using Carnoy-fixative and the ABC-method, we demonstrate light microscopic immunohistochemical localization of peroxisomal antigens in several rat tissues as well as in human post-mortem liver.

[The peroxisomes of eosinophilic leukocytes]. / Peroksidazosomy éozinofil'nykh leikotsitov.

Functional properties of peroxidase-containing granules (peroxidasosomes) of eosinophiles are discussed. Findings are reported on the activities of peroxidase systems and of non-enzymic cationic proteins which occur in the eosinophilic peroxidasosomes in normal state and in various pathological states as well as their antimicrobial and antiparasitic activities.

Antibodies directed against a yeast carboxyl-terminal peroxisomal targeting signal specifically recognize peroxisomal proteins from various yeasts.

The carboxyl-terminal tripeptide Ala-Lys-Ile is essential for targeting Candida tropicalis trifunctional enzyme (hydratase-dehydrogenase-epimerase) to peroxisomes of both Candida albicans and Saccharomyces cerevisiae (Aitchison,J.D., Murray, W.W. and Rachubinski, R. A. (1991).J. Biol. Chem. 266, 23197-23203). We investigated the possibility that this tripeptide may act as a general peroxisomal targeting signal (PTS) for other proteins in the yeasts C. tropicalis, C. albicans, Yarrowia lipolytica and S. cerevisiae, and in rat liver. Anti-AKI antibodies raised against the carboxyl-terminal 12 amino acids of trifunctional enzyme were used to search for this PTS in proteins of these yeasts and of rat liver. The anti-AKI antibodies reacted exclusively with multiple peroxisomal proteins from the yeasts C. tropicalis, C. albicans and Y. lipolytica. There was a weak reaction of the antibodies with one peroxisomal protein from S. cerevisiae and no reaction with peroxisomal proteins from rat liver. Antibodies directed against a synthetic peptide containing a carboxyl-terminal Ser-Lys-Leu PTS (Gould, S. J., Krisans, S., Keller, G.-A. and Subramani, S. (1990). J. Cell Biol. 110,27-34) reacted with multiple peroxisomal proteins of rat liver and with peroxisomal proteins of yeast distinct from those identified with anti-AKI antibodies. These results provide evidence that several peroxisomal proteins of different yeasts contain a PTS antigenically similar to that of C. tropicalis trifunctional enzyme and that this signal is absent from peroxisomal proteins from at least one mammalian system, rat liver.

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats.

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.

A novel 57 kDa peroxisomal membrane polypeptide detected by monoclonal antibody (PXM1a/207B).

BALB/c mice were immunized with peroxisomal membranes prepared from rat liver. Spleen cells were fused with myeloma cells (P3/U1) and the hybridomas were selected using peroxisomal membranes. A monoclonal antibody (PXM1a/207B) which recognized peroxisomal membranes was selected. Using the antibody, a novel 57 kDa polypeptide was identified in the peroxisomal membrane fraction. Immunoblot analysis of the subcellular fractions demonstrated that the 57 kDa polypeptide was present exclusively in peroxisomal membranes. The 57 kDa polypeptide was partially digested by trypsin and chymotrypsin under conditions where peroxisomal particles remained intact, indicating that the polypeptide is exposed to the cytosolic face of the peroxisomal membrane. The amount of 57 kDa polypeptide increased in parallel with proliferation of peroxisomes by administration of clofibrate.

Presence of D-amino-acid oxidase protein in mutant mice lacking D-amino-acid oxidase activity.

1. Mutant mice lacking D-amino-acid oxidase activity were examined as to whether they possessed the enzyme protein. 2. Immunoblotting using an antibody against hog kidney D-amino-acid oxidase showed that kidney homogenates of the mutant mice as well as that of the normal mice had proteins reactive to the antibody. 3. Peroxisomal proteins of the kidney cells of the mutant mice were not different from those of the normal mice. 4. The peroxisomes of the mutant mice possessed a protein reactive to the antibody in the immunoblotting whose size was the same as the D-amino-acid oxidase protein present in the peroxisomes of the normal mice. 5. These results suggest that the mutant mice synthesize the D-amino-acid oxidase protein and integrate it into peroxisomes, though it is a nonfunctional enzyme.

The inhibitory effect of granulocyte factor in mixed lymphocyte reaction.

The polymorphonuclear leukocytes (PMNLs) depress mixed leukocyte reaction (MLR) in vitro. The active factor involved in inhibition was found to be localized in the specific granules of PMNL. The adherent granulocytes secreted granulocyte factor (GF) which also diminished MLR. The functional activity of GF is reflected by inhibition of responding cell proliferation exclusively during induction of MLR; moreover, GF depresses generation of the specific suppressor cells associated with MLR. The effect of GF is modified using specific antisera.

A human autoantibody to peroxisomes.

A complement fixing, non-organ specific IgG autoantibody is described in 29 patients. The autoantibody gives a highly characteristic, granular staining of liver cells, proximal kidney tubules and stomach surface epithelium. By studies with various subcellular fractions from rat liver, employing two different techniques (quantitative complement fixation, and absorption combined with indirect immunofluorescence) the autoantibody was shown to react with a peroxisomal antigen. No convincing clinical correlations were found.