The Sp1 transcription factor is essential for the expression of gliostatin/thymidine phosphorylase in rheumatoid fibroblast-like synoviocytes.

INTRODUCTION: Gliostatin/thymidine phosphorylase (GLS/TP) has angiogenic and arthritogenic activities, and aberrant GLS production has been observed in the active synovial membranes of rheumatoid arthritis (RA) patients. The human GLS gene promoter contains at least seven consensus binding sites for the DNA binding protein Sp1. Here we examined whether Sp1 is necessary for GLS production in RA. We also studied the effects of the Sp1 inhibitor mithramycin on GLS production in RA fibroblast-like synoviocytes (FLSs). METHODS: FLSs from RA patients were treated with specific inhibitors. The gene and protein expression of GLS were studied using the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and an enzyme immunoassay. Intracellular signalling pathway activation was determined by western blotting analysis, a luciferase assay, a chromatin immunoprecipitation (ChIP) assay and a small interfering RNA (siRNA) transfection. RESULTS: The luciferase and ChIP assays showed that Sp1 binding sites in the GLS promoter were essential for GLS messenger RNA (mRNA) expression. GLS production was suppressed in FLSs by siRNA against Sp1 transfection. Mithramycin decreased GLS promoter activity, mRNA and protein expression in FLSs. Tumour necrosis factor-α (TNF-α) significantly increased GLS expression in RA FLSs; this effect was reduced by pre-treatment with cycloheximide and mithramycin. CONCLUSIONS: Pretreatment of mithramycin and Sp1 silencing resulted in a significant suppression of GLS production in TNF-α-stimulated FLSs compared to controls. GLS gene expression enhanced by TNF-α was partly mediated through Sp1. As physiological concentrations of mithramycin can regulate GLS production in RA, mithramycin is a promising candidate for anti-rheumatic therapy.

Ecophysiology of uncultured filamentous anaerobes belonging to the phylum KSB3 that cause bulking in methanogenic granular sludge.

A filamentous bulking of a methanogenic granular sludge caused by uncultured filamentous bacteria of the candidate phylum KSB3 in an upflow anaerobic sludge blanket (UASB) system has been reported. To characterize the physiological traits of the filaments, a polyphasic approach consisting of rRNA-based activity monitoring of the KSB3 filaments using the RNase H method and substrate uptake profiling using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was conducted. On the basis of rRNA-based activity, the monitoring of a full-scale UASB reactor operated continuously revealed that KSB3 cells became active and predominant (up to 54% of the total 16S rRNA) in the sludge when the carbohydrate loading to the system increased. Batch experiments with a short incubation of the sludge with maltose, glucose, fructose, and maltotriose at relatively low concentrations (approximately 0.1 mM) in the presence of yeast extract also showed an increase in KSB3 rRNA levels under anaerobic conditions. MAR-FISH confirmed that the KSB3 cells took up radioisotopic carbons from [(14)C]maltose and [(14)C]glucose under the same incubation conditions in the batch experiments. These results suggest that one of the important ecophysiological characteristics of KSB3 cells in the sludge is carbohydrate degradation in wastewater and that high carbohydrate loadings may trigger an outbreak of KSB3 bacteria, causing sludge bulking in UASB systems.

Comparative analysis of bacterial and archaeal communities in methanogenic sludge granules from upflow anaerobic sludge blanket reactors treating various food-processing, high-strength organic wastewaters.

A comprehensive survey of bacterial and archaeal community structures within granular sludges taken from twelve different types of full-scale, food-processing wastewater-treating, upflow anaerobic sludge blanket (UASB) reactors was performed with a 16S rRNA gene-based clone library method. In total, 1,282 bacterial 16S rRNA gene clones and 722 archaeal clones were analyzed, and their identities were determined by phylogenetic analyses. Overall, clones belonging to the bacterial phyla Proteobacteria (the class Deltaproteobacteria in particular), Firmicutes, Spirochaetes, and Bacteroidetes were observed in abundance within the bacterial clone libraries examined, indicating common bacterial denominators in such treatment systems. Within the domain Archaea, clones affiliated with the classes Methanomicrobia and Methanobacteria were found to be abundant in the archaeal libraries. In relation to features of reactor performance (such as chemical oxygen demand removal, fatty acid accumulation, and sludge bulking), possible representative phylotypes likely to be associated with process failures, such as sludge bulking and the accumulation of propionate, were found in comparative analyses of the distribution of phylotypes in the sludge libraries.