We have isolated an 18-kDa peptide (designated spi8, for 18-kDa secreted protein) from the conditioned medium of lipopolysaccharide-stimulated RAW 264.7 murine macrophages. Purified spi8 had in vivo inflammatory activity and in vitro chemotactic activity for human peripheral blood polymorphonuclear leukocytes and monocytes. Surprisingly, N-terminal sequencing and tryptic mapping studies revealed that spi8 and cyclophilin, an intracellular protein that binds the immunosuppressive drug cyclosporin A, are highly homologous. The in vitro chemotactic activity of spi8 on monocytes was blocked by cyclosporin A but not by cyclosporin H, a structural analog of cyclosporin A that does not bind cyclophilin. Like purified porcine cyclophilin, mouse spi8 exhibited peptidyl-prolyl cis-trans isomerase activity. Medium conditioned by lipopolysaccharide-stimulated resident peritoneal exudate macrophages isolated from C57BL/6 mice contained substantially higher levels of sp18/cyclophilin than medium conditioned by nonstimulated macrophages. The observation that spl8/cyclophilin exhibits proinflammatory activity and is secreted by macrophages in response to endotoxin suggests that this protein may function as a cytokine, and invites the hypothesis that the immunosuppressive action of cyclosporin A results in part from interaction with an extracellular form of cyclophilin released as a mediator of immune and inflammatory functions.
Sedimentable hydrogenase activity was demonstrated in cell-free extracts from both zoospores and vegetative growth of the anaerobic rumen fungus Neocallimastix patriciarum. Electron micrographs of the fraction enriched in hydrogenase activity contained finely granular microbody-like organelles, about 0.5 micron in diameter and having an equilibrium density of about 1.2 g X ml-1 in sucrose, 1.12 g X ml-1 in Percoll and 1.27-1.28 g X ml-1 in Metrizamide. These organelles, which are sedimentable at 10(5) g-min, bear no similarity to mitochondria, but are morphologically similar to hydrogen-evolving organelles possessed by certain anaerobic protozoa and termed 'hydrogenosomes'. Other typical hydrogenosomal enzymes, namely 'malic' enzyme, pyruvate:ferredoxin oxidoreductase and NADPH:ferredoxin oxidoreductase, were enriched in the same particle fraction as hydrogenase. The synthesis of pyruvate:ferredoxin oxidoreductase was found to be suppressed when the organism was cultured under an atmosphere of CO2, and an alternative pathway is proposed for growth under these conditions.
This paper reports for the first time the presence in the anaerobic rumen ciliate Dasytricha ruminantium (Schuberg) of microbody-like organelles, about 0.5 micrometer diameter, with a granular matrix and an equilibrium density of approx. 1.18 g/ml. These organelles can be isolated in a fraction sedimented at 10(5) g-min that contains 67% of the total pyruvate synthase (EC 1.2.7.1), 66% of the hydrogenase (EC 1.18.3.1) and 20% of the lactate dehydrogenase (EC 1.1.1.27). Thus in several respects this fraction is enzymically similar to those containing hydrogenosomes in some other parasitic anaerobic protozoa (the trichomonads). However, in contrast with the hydrogenosomes of trichomonads, the oxygen-tolerant enzyme malate dehydrogenase (decarboxylating) (EC 1.1.1.40) is not particulate, but occurs only in the cytosol. These results enable the proposal of a scheme for the pathway of product formation (acetate, lactate, CO2 and H2) from carbohydrates.
Cia5 is a locus on rat chromosome 10 which regulates the severity of collagen- and pristane-induced arthritis (CIA and PIA). To refine the region toward positional identification, Cia5 subcongenic strains were generated and studied in PIA and CIA. The protective effect of the telomeric locus Cia5a was confirmed in both models. A second arthritis severity locus (Cia5d) was identified within the most centromeric portion of Cia5. DA.F344(Cia5d) rats had a significantly lower median arthritis severity index in PIA, but not in CIA, compared with DA. On histologic analyses DA.F344(Cia5a) and DA.F344(Cia5d) congenics with PIA preserved a nearly normal joint architecture compared with DA, including significant reduction in synovial hyperplasia, pannus, angiogenesis, inflammatory infiltration, bone and cartilage erosions. Cia5 and Cia5a synovial levels of IL-1β mRNA were reduced. Although both DA.F344(Cia5) and DA.F344(Cia5a) rats were protected in CIA, the arthritis scores of DA.F344(Cia5) were significantly higher than those of DA.F344(Cia5a), suggesting the existence of a third locus where F344-derived alleles centromeric from Cia5a contribute to increased arthritis severity. The existence of the third locus was further supported by higher levels of autoantibodies against rat type II collagen in DA.F344(Cia5) congenics compared with DA.F344(Cia5a). Our results determined that Cia5 contains three major arthritis severity regulatory loci regulating central events in the pathogenesis of arthritis, and differentially influencing CIA and PIA. These loci are syntenic to regions on human chromosomes 17q and 5q implicated in the susceptibility to rheumatoid arthritis, suggesting that the identification of these genes will be relevant to human disease.
Objective. To construct rats congenic for the chromosome 2 arthritis-regulatory quantitative trait locus Cia10, originally identified in a (DA ؋ ACI)F 2 intercross rat strain that had been assessed for collagen-induced arthritis (CIA), and to determine the effect of this congenic interval on arthritis severity, joint histologic structure, and cytokine transcription in rats with pristane-induced arthritis (PIA).Methods. A 52.6-MB interval derived from the ACI (CIA-and PIA-resistant) strain and containing the Cia10 interval was introgressed into the DA (arthritissusceptible) background through genotype-guided congenic breeding. Homozygous male and female DA.ACI(Cia10) congenic rats were studied for their susceptibility to and severity of PIA, and were compared with same-sex DA rats. Histologic analyses were done on hind paws collected on day 32 following the pristane injection. Levels of interleukin-1 (IL-1) and tumor necrosis factor ␣ (TNF␣) messenger RNA (mRNA) were measured with real-time polymerase chain reaction on synovial tissues from day-32 ankles.Results. Both male and female DA.ACI(Cia10) congenic rats developed a significantly milder form of arthritis, with a 95% and 92% reduction in the arthritis severity index compared with DA male and female controls, respectively (males P < 0.001 and females P ؍ 0.003). DA.ACI(Cia10) congenic rat synovial tissue was more likely to preserve its normal histologic architecture, including minimal to no cartilage and bone erosions, synovial hyperplasia, and pannus formation, and reduced numbers of vessels (angiogenesis), when compared with DA synovial tissue. There was a 2.7-and 2.4-fold reduction in the amount of IL-1 and TNF␣ mRNA, respectively, in the synovial tissue of DA.ACI-(Cia10) congenic rats compared with DA rats. Sequencing analyses of complementary DNA for the Cia10-predicted candidate gene Ptpn8, the rat homolog of the rheumatoid arthritis (RA)-susceptibility gene PTPN22, revealed no polymorphisms between the DA and ACI strains.Conclusion. This study determined that Cia10 harbors a major autoimmune arthritis-regulatory gene. This gene regulates clinical disease severity, histologic damage, and the levels of at least two central proinflammatory cytokines. We are in the process of narrowing down the critical region for positional cloning of the Cia10 gene. The identification of this gene will provide novel targets or pathways for focused candidate-gene studies in RA.
A mitochondrion-like organelle (MLO) was isolated from isotonic homogenates of Blastocystis. The organelle sedimented at 5000 g for 10 min, and had an isopycnic density in sucrose of 1.2 g ml "1. Biochemical characterization enabled the demonstration of several key enzymes that allowed the construction of a metabolic pathway consisting of an incomplete Krebs cycle linked to the oxygen-sensitive enzymes pyruvate : NADP + oxidoreductase (PNO), acetate : succinate CoA transferase (ASCT) and succinate thiokinase (STK), which cumulatively are responsible for recycling CoA and generating ATP. The organelle differs from typical aerobic mitochondria in possessing an oxygen-sensitive PNO that can use FAD + or FMN + as electron acceptor but is inactive with NAD + , Spinacia oleracea ferredoxin or Clostridium pasteurianum ferredoxin. A gene with 77 % sequence similarity to the PNO mitochondrion precursor cluster from Euglena gracilis sp[Q941N5] was identified in the Blastocystis genome database. A second cluster with 56 % sequence similarity to the pyruvate : ferredoxin oxidoreductase (PFOR) from Trichomonas vaginalis was also identified, which is in agreement with the concept that the PNO gene arose through the fusion of a eubacterial gene for PFOR with the gene for NADPH : cytochrome p450 reductase. Hydrogenase activity was not detected under the conditions used in this study. The Blastocystis oranelle therefore demonstrates significant biochemical differences from traditional mitochondria and hydrogenosomes, but possesses features of both. Based upon the results of this study, the Blastocystis organelle falls into the category of a MLO.
Neocallimastix patriciarum was successfully frozen in liquid media containing 5% dimethylsuphoxide as cryoprotectant. Initial freezing and storage for 24 h in solid CO2 before transfer to liquid N2 was necessary for survival. Long‐term storage under liquid N2, solid CO2 and at ‐ 80°C was tested; only storage in liquid N2 proved successful for periods in excess of several months. Analysis of metabolic end‐products and electron microscopy indicated that no morphological or biochemical changes had occurred in cultures preserved for 1 year. Piromonas communis and a recent isolate from the ovine rumen were preserved under the same conditions. The method does not require controlled freezing equipment, making it an easily applied laboratory technique.
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