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Osteoarthritis (OA) is a multifactorial degenerative pathology, whose progression is exacerbated by pro-inflammatory cytokines signaling. Among the changes triggered in chondrocytes during inflammation, modified expression of tiny epigenetic regulators as microRNAs was shown having deleterious implications for articular cartilage. Aim of the present study was to identify differentially expressed microRNAs in human OA cartilage and to determine their relevance to pathological progression. An OA model based on inflammatory stimulation of a chondrocytic human cell line was used to analyze microRNAs deregulation, and results revealed miR-149 severely down-regulated by IL1β and TNFα. Real-time PCR analysis of miR-149 was exerted also in human primary chondrocytes isolated from cartilage of OA donors and postmortem from subjects with no known history of OA, confirming down-regulation in osteoarthritis. Moving on a functional study, miR-149 regulatory effect on tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL1β) and interleukin 6 (IL6) 3'UTRs was evaluated by luciferase assays, and chondrocytes production of TNFα upon miR-149 transfection was measured by enzyme-linked immuno sorbent assay. We found that miR-149 is down-regulated in OA chondrocytes, and this decrease seems to be correlated to increased expression of pro-inflammatory cytokines such as TNFα, IL1β and IL6. OA is a multifactorial disease and we think that our results give new insights for understanding the complex mechanisms of osteoarthritic pathogenesis.

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The hyperthermophilic archaebacterium Pyrococcus furiosus contains high levels of NAD(P)-dependent glutamate dehydrogenase activity. The enzyme could be involved in the first step of nitrogen metabolism, catalyzing the conversion of 2-oxoglutarate and ammonia to glutamate. The enzyme, purified to homogeneity, is a hexamer of 290 kDa (subunit mass 48 kDa). Isoelectric-focusing analysis of the purified enzyme showed a pl of 4.5. The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate but utilizes both NADH and NADPH as cofactors. The purified enzyme reveals an outstanding thermal stability (the half-life for thermal inactivation at 100°C was 12 h), totally independent of enzyme concentration.P. furiosus glutamate dehydrogenase represents 20% of the total protein; this elevated concentration raises questions about the roles of this enzyme in the metabolism of P..furiosus.Recently we reported the purification of an NAD(P)-dependent glutamate dehydrogenase from the thermophilic archaebacterium Sulfolobus so@ztaricus [l, 21. The enzyme of this microorganism is probably involved in the first step of ammonia assimilation by the following reaction: glutamate + NAD(P)+ + H 2 0 + 2-oxoglutarate + NH,' + NAD(P)H + H + .The glutamate dehydrogenase from S . solfutaricus presents some interesting properties; it has a double coenzyme specificity, is strictly specific for L-glutamate and 2-oxoglutarate and its thermal stability is a function of the enzyme concentration [2]. This latter property is in good agreement with the observed self-associating behaviour of the purified enzyme.Among archaebacterial glutamate dehydrogenases, the only data available for comparison are restricted to the halophilic phenotype [3]. Therefore, it is difficult to extrapolate from our observations on S. solfituricus glutamate dehydrogenase to obtain general rules for thermal adaptation in this class of enzymes and general knowledge regarding nitrogen metabolism in archaebacteria.To provide more information about the enzymological properties of archaebacterial glutamate dehydrogenases and to extend our investigations towards more thermostable forms of this enzyme, we attampted to purify glutamate dehydrogenase from the so-called 'hyperthermophiles'. These remarkable microorganisms, recently discovered, grow optimally at temperatures near 100 "C [4]. They all are archaebacteria and most of them are strict anaerobes and depend on the reduction of elemental sulfur for growth. So far, hyperthermophiles -___ have been classified into three distinct genera, Pyrodiictium, Pyrobaculurn and Pyrococcus. As a source of hyperthermophilic glutamate dehydrogenase we used Pyrococcus furiosus [5], one of the most interesting members of this last genus because of its relatively high cell yield and rapid growth rate. The microorganism grows optimally at 10O'C by a fermentative-type metabolism and is a strict heterotroph which utilizes both simple and complex carbohydrates and produces only H2 and C 0 2 as detectable products. P. furiosus reduces eleme...

The aim of this pilot study was to analyze the effects of glucosamine (GlcN) and its N-acetyl-phenylalanine derivative (NAPA) in Vitamin A model of osteoarthritis (OA) in rabbits. GlcN or NAPA or saline solution was intra-articularly administered in rabbit OA knees. Histological analysis revealed that treatment with GlcN or NAPA was associated with more homogeneous chondrocyte cellularity, absence of fissures and fragmentation and more intense staining of the matrix with Alcian Blue compared to the articular surfaces of the knees treated with saline solution. Comparative in vitro study performed on rabbit primary chondrocytes revealed that GlcN and NAPA were also able to counteract the IL-1beta-upregulation of genes coding for metalloproteases and inflammatory cytokines. Our preliminary in vivo and in vitro studies suggest that GlcN and NAPA could play a disease-modifying protective role in OA by an anti-catabolic effect and an anti-inflammatory activity on chondrocytes.

IntroductionNuclear factor-κB (NF-κB) transcription factor regulates several cell signaling pathways, such as differentiation and inflammation, which are both altered in osteoarthritis. Inhibitor κB kinase (IKK)α and IKKβ are kinases involved in the activation of the NF-κB transcription factor. The aim of the present study was to determine the effects of glucosamine (GlcN), which is administered in the treatment of osteoarthritis, and of its 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA) derivative on IKK kinases and, consequently, on NF-κB activation in human chondrocytes.MethodsThe human chondrosarcoma cell line HTB-94 and human primary chondrocytes were stimulated with tumor necrosis factor (TNF)α after pre-treatment with GlcN or NAPA. Gene mRNA expression level was evaluated by real-time PCR. Inhibitor κB protein (IκB)α phosphorylation and p65 nuclear re-localization were analyzed by Western blotting; IKKα nuclear re-localization was also investigated by immunocytochemistry and Western blotting. IKK kinase activity was studied by in vitro kinase assay.ResultsAfter TNFα stimulation, the mRNA expression level of some of the genes under NF-κB control, such as interleukin (IL)-6 and IL-8, increased, while treatment with GlcN and NAPA reverted the effect. We investigated the possibility that GlcN and NAPA inhibit IKK kinase activity and found that NAPA inhibits the IKKα kinase activity, whereas GlcN does not. Interestingly, both GlcN and NAPA inhibit IKKα nuclear re-localization.ConclusionsOur results demonstrate that glucosamine and its peptidyl derivative can interfere with NF-κB signaling pathway by inhibiting IKKα activity in human chondrocytes. However, the mechanism of action of the two molecules is not completely overlapping. While NAPA can both specifically inhibit the IKKα kinase activity and IKKα nuclear re-localization, GlcN only acts on IKKα nuclear re-localization.

An NAD(P)-dependent glutamate dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Suljolobus soljataricus. The enzyme is a hexamer (subunit mass 45 kDa) which dissociates into lower states of association when submitted to gel filtration. lsoelectric focusing analysis of the purified enzyme showed a PI of 5.7 and occasionally revealed microheterogeneity. The enzyme is strictly specific for the natural substrates 2-oxoglutarate and L-glutamate, but is active with both NADH and NADPH. S. solfataricus glutamate dehydrogenase revealed a high degree of thermal stability (at 80°C the half-life was 15 h) which was strictly dependent on the protein concentration. Very high levels of glutamate dehydrogenase were found in this archaebacterium which suggests that the conversion of 2-oxoglutarate and ammonia to glutamate is of central importance to the nitrogen metabolism in this bacterium.