Detection of oncofetal h19 RNA in rheumatoid arthritis synovial tissue.

The expression of oncofetal H19 RNA and its localization/cellular source was analyzed in synovial tissue (ST) and isolated synovial macrophages (Mphi) or synovial fibroblasts (SFBs) by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR showed significantly higher H19 expression in ST from patients with rheumatoid arthritis (RA) (P = 0.000) and osteoarthritis (OA) (P = 0.009) than in normal/joint trauma controls (N/JT), but comparable levels in reactive arthritis. In situ hybridization demonstrated strong signals in all RA-ST samples (n = 8), with > or =85% positive cells in the lining layer, diffuse infiltrates, and stroma regions. In lymphoid aggregates and endothelial cells only 20% were positive. RA-ST contained a significantly higher percentage of strongly positive lining cells than OA-ST and N/JT-ST. H19 RNA was expressed in both Mphi and SFBs, as confirmed by RT-PCR in isolated RA Mphi and SFBs (n = 3). In RA-SFBs, low constitutive H19 RNA expression in culture (10% fetal calf serum) was strongly increased on starvation (3.5-fold, 1% fetal calf serum), with or without the addition of interleukin-1beta (10 to 100 U/ml), tumor necrosis factor-alpha (1 to 25 ng/ml), or platelet-derived growth factor-BB (2.5 to 10 U/ml). In OA-SFBs, this starvation-induced increase was lower (twofold), reaching significant differences compared with RA-SFBs after stimulation with interleukin-1beta and platelet-derived growth factor-BB. In both RA- and OA-SFBs, the MAP-kinase ERK-1/2 pathway and the phosphatidylinositol-3 kinase pathway influenced H19 RNA expression, as shown by inhibitor studies. Significant overexpression of H19 RNA and its increased sensitivity to starvation/cytokine regulation in RA suggests a pathogenetic role of this oncofetal gene, possibly reflecting embryonal dedifferentiation of the adult ST and/or ongoing inflammatory/oxidative stress.

Effects of lipopolysaccharide extracted from Prevotella intermedia on bone formation and on the release of osteolytic mediators by fetal mouse osteoblasts in vitro.

Prevotella intermedia, a Gram-negative obligate anaerobic black-pigmented oral bacterium, belongs to a small group of microorganisms that is closely associated with the initiation of periodontal diseases. Lipopolysaccharide (LPS), an outer membrane component, is one of the main virulence factors of this bacterium. The aim of this study was to examine the effects of Prev. intermedia lipopolysaccharide, extracted by the hot-phenol-water method, on differentiation (alkaline phosphatase activity) and mineralisation (calcium incorporation) of fetal mouse calvarial cells in vitro and to determine the release of the important osteolytic factors nitric oxide, interleukin-6 (IL-6) and matrix metalloproteinases by these cells after treatment with different concentrations of Prev. intermedia lipopolysaccharide (0.2-25 microg/ml). By gelatin zymography, we also characterized the matrix metalloproteinases released by these osteoblasts. Treatment with Prev. intermedia lipopolysaccharide dose-dependently inhibited bone formation by reducing alkaline phosphatase activity and calcium incorporation and induced the release of nitric oxide, IL-6 and the latent proforms of MMP-2 and MMP-9 by fetal mouse osteoblasts in organoid culture. These results indicate that the lipopolysaccharide from Prev. intermedia not only participates in periodontal tissue destruction and alveolar bone resorption, but also inhibits bone formation.

Sugar-mediated lattice contacts in crystals of a plant glycoprotein.

Pokeweed antiviral protein, PAP-S(aci), isolated from seeds of the Chinese pokeweed plant, Phytolacca acinosa, belongs to the family of type-1 ribosome-inactivating proteins (RIPs). Type-1 RIPs are approximately 30-kDa N-glycosidases that inactivate eukaryotic and prokaryotic ribosomes via a site-specific depurination of ribosomal RNA (rRNA). Here we describe the preliminary X-ray structure determination at 1.7 A resolution of one PAP isoenzyme from seeds, PAP-S1(aci), after crystallisation from a heterogeneous mixture of two isoenzymes. PAP-S1(aci) possesses a rare type of glycosylation, specifically, N-linked N-acetyl-D-glucosamine monosaccharide (GlcNAc) substitutions at canonical Asn-Xaa-Ser/Thr sequons. One GlcNAc residue was found to play a critical role in crystal lattice formation, forming a packing interface across a crystallographic two-fold with the identical sequon of an adjacent monomer. This observation suggests that deglycosylation protocols for the crystallisation of glycoproteins should be designed to allow for exploitation of the crystal packing potential of the innermost core sugar residue (N-linked GlcNAc or O-linked GalNAc).

Structural studies of an eukaryotic cambialistic superoxide dismutase purified from the mature seeds of camphor tree.

An iron-superoxide dismutase (SOD) was purified and characterized from the mature seeds of camphor tree (Cinnamomum camphora). The ultraviolet and visible absorption spectra of camphor Fe-SOD showed patterns typical of cambialistic Fe-SODs. The inductively coupled plasma assay indicated that there was 0.5-1 atom of Fe(2+) per camphor Fe-SOD subunit. The cDNA of camphor Fe-SOD, including the coding region and the 3' noncoding region, was obtained by reverse transcription polymerase chain reaction using the total RNA from immature seeds of C. camphora as template and then sequenced. The complete amino acid sequence of camphor Fe-SOD was deduced from the cDNA sequence. The correctness of the amino acid sequence was confirmed by directly sequencing five peptide fragments of the enzyme. The molecular mass calculated for the camphor Fe-SOD subunit from its 204 amino acid residues was 22,930.6 Da, The cDNA of camphor Fe-SOD was cloned into the expression vector PMFT7-5 and then expressed in Escherichia coli strain BL21. The reconstructed Fe- or Mn-SOD was purified to homogeneity through column chromatography. Activity of the Fe- or Mn-SOD was found to be almost equal to that of natural camphor Fe-SOD, which is the first cambialistic SOD isolated from eukaryotic cells.