Oxygen radical generation by isolated microsomes from soybean seedlings

The generation of active oxygen species by microsomes isolated from soybean seedlings was studied. NADPH-dependent superoxide anion production was 5.0 ± 0.4 nmol · min⁻¹ mg⁻¹ of microsomal protein. Hydrogen peroxide generation by microsomes was 1.40 ± 0.05 nmol · min⁻¹ mg⁻¹ of protein. Hydroxyl radical production, in the presence of ferric EDTA, evaluated through the generation of formaldehyde from dimethyl sulfoxide or tert-butyl alcohol was 0.50 ± 0.04 and 0.44 ± 0.03 nmol · min⁻¹ mg⁻¹, respectively. NADH proved to be suitable as cofactor for oxygen radical generation by microsomes from soybean seedlings. Because transition metals are implicated in radical generation by biological systems, the ability of microsomal membranes to reduce iron complexes was studied. Ferric ATP, ferric citrate, ferric ADP, ferric diethylenetriamine pentaacetic acid, and ferric EDTA were efficiently reduced in the presence of either NADPH or NADH as cofactor. The pattern of effectiveness of the different ferric complexes, on superoxide anion, hydrogen peroxide, and hydroxyl radical production, was similar to that found with animal microsomes. The data presented here indicate that microsomal ability to catalyze oxygen radical generation must be considered as an important contribution to cellular radical steady-state concentrations in cells from soybean seedlings.     

Recombinant production and biochemical characterization of a hyperthermostable α-glucan/maltodextrin phosphorylase from Pyrococcusfuriosus

Alpha-glucan phosphorylase catalyzes the reversible cleavage of α-1-4-linked glucose polymers into α-D-glucose-1-phosphate. We report the recombinant production of an α-glucan/maltodextrin phosphorylase (PF1535) from a hyperthermophilic archaeon, Pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. The apparent 98 kDa recombinant enzyme was active over a broad range of temperatures and pH, with optimal activity at 80 °C and pH 6.5–7. This archaeal protein retained its complete activity after 24 h at 80 °C in Tris-HCl buffer. Unlike other previously reported phosphorylases, the Ni-affinity column purified enzyme showed broad substrate specificity in both the synthesis and degradation of maltooligosaccharides. In the synthetic direction of the enzymatic reaction, the lowest oligosaccharide required for the chain elongation was maltose. In the degradative direction, the archaeal enzyme can produce glucose-1-phosphate from maltotriose or longer maltooligosaccharides including both glycogen and starch. The specific activity of the enzyme at 80 °C in the presence of 10 mM maltoheptaose and at 10 mg ml^–1 glycogen concentration was 52 U mg^–1 and 31 U mg^–1, respectively. The apparent Michaelis constant and maximum velocity for inorganic phosphate were 31 ± 2 mM and 0.60 ± 0.02 mM min^–1 µg^–1, respectively. An initial velocity study of the enzymatic reaction indicated a sequential bi-bi catalytic mechanism. Unlike the more widely studied mammalian glycogen phosphorylase, the Pyrococcus enzyme is active in the absence of added AMP.     

Fluorimetric determination of monobromobimane and o-phthalaldehyde adducts of γ-glutamylcysteine and glutathione: application to assay of γ-glutamylcysteinyl synthetase activity and glutathione concentration in liver

The reversed-phase HPLC separation of fluorescent o-phthalaldehyde (OPA) derivatives has been applied to the assay of hepatic γ-glutamylcysteine and glutathione (GSH) levels and the enzymes producing these peptides. The method has been compared to the assay using monobromobimane (MB) as the derivatizing agent. The OPA method has the advantage of faster derivitization, the lack of need to adjust the pH, isocratic separation and selectivity for GSH and γ-glutamylcysteine. The MB method requires pH adjustment following derivatization and gradient elution chromatography. MB is also non-selective, yielding fluorescent derivatives of all biological thiols and more interfering peaks on the chromatogram. MB-based analyses are also approximately sixty times more expensive per sample. MB yields fluorescent degradation products on exposure to light. OPA adducts are stable for up to ten days when stored at −20°C. OPA detection is sensitive to 12.5 pmol in the sample, at a signal-to-noise ratio of 2.5. The two methods correlate well. Hepatic γ-glutamylcysteine synthetase in the same liver preparation was found to be 4.85±0.47 nmol min⁻¹ mg⁻¹ protein by the OPA method and 4.42±0.52 nmol min⁻¹ mg⁻¹ protein by the MB method. GSH concentrations were found to be 90.4±6.5 nmol/mg protein for the OPA method and 92.5±3.4 for the MB method.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Anaerobic degradation of phenol via carboxylation to 4-hydroxybenzoate: in vitro study of isotope exchange between 14CO2 and 4-hydroxybenzoate

Extracts of denitrifying bacteria grown anaerobically with phenol and nitrate catalyzed an isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate. This exchange reaction is ascribed to a novel enzyme, phenol carboxylase, initiating the anaerobic degradation of phenol by para-carboxylation to 4-hydroxybenzoate. Some properties of this enzyme were determined by studying the isotope exchange reaction. Phenol carboxylase was rapidly inactivated by oxygen; strictly anoxic conditions were essential for preserving enzyme activity. The exchange reaction specifically was catalyzed with 4-hydroxybenzoate but not with other aromatic acids. Only the carboxyl group was exchanged; [U-¹⁴C]phenol was not exchanged with the aromatic ring of 4-hydroxybenzoate. Exchange activity depended on Mn²⁺ and inorganic phosphate and was not inhibited by avidin. Ortho-phosphate could not be substituted by organic phosphates nor by inorganic anions; arsenate had no effect. The pH optimum was between pH 6.5–7.0. The specific activity was 100 nmol ¹⁴CO₂ exchange · min^-1 · mg^-1 protein. Phenol grown cells contained 4-hydroxybenzoyl CoA synthetase activity (40 nmol · min^-1 · mg^-1 protein). The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed.     

Desiccation induced changes in osmolytes production and the antioxidative defence in the cyanobacterium Anabaena sp. PCC 7120

Cells of Anabaena sp. PCC 7120, a low desiccation tolerant cyanobacterium, was subjected to prolonged desiccation and effect of loss of water was examined on production of osmolytes, and antioxidant response as well as on overall viability in terms of photosynthetic activity. During dehydration (22 h), the organism maintained about 98.5 % loss of cellular water, yet cells remained viable as about 30 % of photosynthetic O₂-evolution activity resumed upon hydrating (1 h) such cells. In desiccated state, cyanobacterial cells accumulated osmolytes within 1 h though their contents decreased thereafter. The highest levels of trehalose (179 nmol mg⁻¹ protein), sucrose (805 nmol mg⁻¹ protein) and proline (23.2 nmol mg⁻¹ protein) were attained within 1 h. Chlorophyll a and carotenoid contents also increased within 1 h but phycocyanin level showed opposite trend. The oxygen-evolving activity declined in desiccated cyanobacterial biomass while rehydration led to instant recovery, indicating that cells protect the photosynthetic machinery against desiccation. Notwithstanding, activities of antioxidant enzymes (catalase, peroxidase and superoxide dismutase) attained their peaks after 3 h of desiccation, though within 10 min of rehydration, their levels returned back close to basal activities of the cultured cells. We propose that onset of osmolyte production in conjunction with upshift of antioxidant enzymes apparently protects the cyanobacterial cells from desiccation stress.     

Glutamine uptake inZymomonas mobilis

Glutamine was transported inZymomonas mobilis by a mechanism following Michaelis-Menten kinetics with a Km value for glutamine 8 x 10^–5 M and a Vmax value of 15.4 nmol.mg^–1, min^–1 or 40 nmol.mg^–1.min^–1 for cells growing on complete medium or minimal medium respectively. The transport of glutamine was energy-dependent and more or less specific for glutamine when cell were grown on rich media. Evidence provided via spheroplasts suggests the possible involvement of a periplasmic component in this transport system.     

A comparison of proline, thiol levels and GAPDH activity in cyanobacteria of different origins facing temperature-stress

Three cyanobacterial strains originating from different habitats were subjected to temperature shift exposures and monitored for levels of proline, thiol and activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Thermophile Mastigocladus laminosus (growth optimum, 40 °C), raised the proline level 4.2-fold at low temperature (20 °C), for the psychrophile Nostoc 593 (growth optimum, 20 °C), it was raised 8-fold at 40 °C while in the mesophile Nostoc muscorum (growth optimum, 30 °C), the imino acid level increased 2.3-fold during temperature shiftdown to 20 °C or 3.5-fold in sets facing shiftup (40 °C). Alterations in thiol levels in the above strains were in line with proline. It is suggested that such fluctuations reflect metabolic shifts as a response to stress. Interestingly, GAPDH activity was maximum at the respective growth temperature optimum of M. laminosus (122 nmol NADPH oxidized min ^–1 mg ^–1 protein) and Nostoc 593 (141 nmol NADPH oxidized min ^–1 mg ^–1 protein) while in N. muscorum, it increased at 40 °C (101 nmol NADPH oxidized min ^–1 mg ^–1 protein) and to 93.3 nmol NADPH oxidized min ^–1 mg ^–1 protein (20 °C) relative to 86 nmol NADPH oxidized min ^–1 mg ^–1 protein at 30 °C. It seems that extremophiles maintain the GAPDH activity/level during growth at their respective temperatures optimal while the mesophile increases it in order to cope up with temperature-stress.     

Identification and Characterization of a Mucilaginibacter sp. Strain QM49 β-Glucosidase and Its Use in the Production of the Pharmaceutically Active Minor Ginsenosides (S)-Rh1 and (S)-Rg2

Here, we isolated and characterized a new ginsenoside-transforming β-glucosidase (BglQM) from Mucilaginibacter sp. strain QM49 that shows biotransformation activity for various major ginsenosides. The gene responsible for this activity, bglQM, consists of 2,346 bp and is predicted to encode 781 amino acid residues. This enzyme has a molecular mass of 85.6 kDa. Sequence analysis of BglQM revealed that it could be classified into glycoside hydrolase family 3. The enzyme was overexpressed in Escherichia coli BL21(DE3) using a maltose binding protein (MBP)-fused pMAL-c2x vector system containing the tobacco etch virus (TEV) proteolytic cleavage site. Overexpressed recombinant BglQM could efficiently transform the protopanaxatriol-type ginsenosides Re and Rg₁ into (S)-Rg₂ and (S)-Rh₁, respectively, by hydrolyzing one glucose moiety attached to the C-20 position at pH 8.0 and 30°C. The K_m values for p-nitrophenyl-β-d-glucopyranoside, Re, and Rg₁ were 37.0 ± 0.4 μM and 3.22 ± 0.15 and 1.48 ± 0.09 mM, respectively, and the V_max values were 33.4 ± 0.6 μmol min⁻¹ mg⁻¹ of protein and 19.2 ± 0.2 and 28.8 ± 0.27 nmol min⁻¹ mg⁻¹ of protein, respectively. A crude protopanaxatriol-type ginsenoside mixture (PPTGM) was treated with BglQM, followed by silica column purification, to produce (S)-Rh₁ and (S)-Rg₂ at chromatographic purities of 98% ± 0.5% and 97% ± 1.2%, respectively. This is the first report of gram-scale production of (S)-Rh₁ and (S)-Rg₂ from PPTGM using a novel ginsenoside-transforming β-glucosidase of glycoside hydrolase family 3.     

Increase of xylan synthetase activity during xylem differentiation of the vascular cambium of sycamore and poplar trees

The activity of a -(1-4)-xylan synthetase, a membrane-bound enzymic system, was measured in particulate enzymic preparations (1,000 g and 1,000–100,000 g pellets) obtained from homogenates of cambial cells, differentiating xylem cells and differentiated xylem cells isolated from actively growing trees of sycamore (Acer pseudoplatamus) and poplar (Populus robusta). The specific activity (nmol of xylan formed min^–1 mg^–1 of protein) as well as the activity calculated on a per cell basis (nmol of xylan formed min^–1 cell^–1) of this enzymic system, markedly increased as cells differentiate from the vascular cambium to xylem. This increase is closely correlated with the enhanced deposition of xylan occurring during the formation of secondary thickening. The possible control of xylan synthesis during the biogenesis of plant cell wall is discussed.     

Sinomenine, theophylline, cysteine, and levamisole: Comparisons of their kinetic effects on mineral formation induced by matrix vesicles

The effects of sinomenine (SIN, an alkaloid extracted from the Chinese medicinal plant Sinomenium acutum used for centuries to treat rheumatic disease, including rheumatoid arthritis) on apatitic nucleation and matrix vesicle (MV)-induced mineral formation were compared with those of cysteine, levamisole, and theophylline. We found that SIN was not an inhibitor of tissue non-specific alkaline phosphatase (TNAP), a marker of biological mineralization, but confirmed that cysteine, levamisole, and theophylline were. Further, none of these four molecules directly affected the nucleation of hydroxyapatite (HA) formation, in contrast to pyrophosphate (PP_i) which did. Incubation of 0.25-1.0 mM cysteine, theophylline, or levamisole with MVs in synthetic cartilage lymph (SCL) containing AMP and Ca²⁺, but not inorganic phosphate (P_i), prolonged the induction time of mineral formation, apparently by inhibiting TNAP activity. SIN at the same levels neither inhibited TNAP activity nor affected the induction time of MV mineral formation. However, SIN did markedly delay MV-induced mineral formation in SCL containing P_i (instead of AMP) in a manner similar to theophylline, but to a lesser extent than levamisole. Cysteine did not delay, in fact it slightly accelerated MV-induced mineral formation in Pi-containing SCL. These findings suggest that levamisole, SIN and theophylline may directly affect Ca²⁺ and/or P_i accretion during mineral formation; however, TNAP was not directly involved. The possible roles of annexins and other ion transporters, such as proteins of the solute carrier family implicated in Ca²⁺ and P_i influx are discussed.     

Identification of a sucrose nonfermenting-1-related protein kinase in sugar beet (Beta vulgaris L.)

A 154 bp polymerase chain reaction product, SBKIN154, showing 76–83% sequence identity with sucrose nonfermenting-1 (SNF1)-related protein kinase nucleotide sequences from other plant species was amplified from sugar beet storage root RNA. Southern blot analysis using SBKIN154 as a hybridisation probe suggested that sugar beet contains either a single-copy SNF1-related gene or a small gene family of highly conserved genes. An antibody raised to a heterologously-expressed fusion of the rye SNF1-related protein kinase, RKIN1, and maltose binding protein, recognised a protein of the expected size (M_r approx. 60,000) on western blots of storage root, stalk, leaf and root extracts. Measurements of SNF1-related activity were made using a specific peptide (SAMS) phosphorylation assay. Activity was highest (0.38 nmol min^-1 mg^-1 protein) in developing storage roots and lowest (0.035 nmol min^-1 mg^-1) in fibrous roots.     

Extracellular ATP Released by Osteoblasts Is A Key Local Inhibitor of Bone Mineralisation

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PP_i). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2P_i). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PP_i-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation.     

Isolation of a highly active H+-ATPase from beef heart mitochondria

The lysolecithin extraction procedure originally described by Sadleret al. (1974) has been modified to yield a H⁺-ATPase with high levels of P_i-ATP exchange activity (400–600 nmol × min^–1 × mg^–1). This activity is further enhanced (1400–1600 nmol × min^–1 × mg^–1) following sucrose density gradient centrifugation in the presence of asolectin. This enhancement results in part from a lipid-dependent activation and in part from removal of inactive complexes. The H⁺ translocating activity of the complex has been determined spectrophotometrically using binding of oxonol VI as an indicator of membrane potential. P_i-ATP exchange, ATP hydrolysis, and oxonol binding are sensitive to energy-transfer inhibitors (oligomycin, rutamycin) and/or uncouplers (DNP, FCCP).     

Leaf developmental stage and tissue location affect the detection of β-glucuronidase in transgenic tobacco plants

Expression in Nicotiana tabaccum L. plants containing the -glucuronidase (GUS) gene under the control of the 35S (CaMV promoter) was affected by tissue type and ontogenic development of the leaves. GUS activity in ontogenetically younger leaves was 1003–1022 nmol 7-hydroxy-4-methylcoumarin (MU) formed mg^–1 (protein) min^–1 and in ontogenetically older leaves was only 140–198 nmol (MU) mg^–1 (protein) min^–1.     

Antiosteoporotic activity of phenolic compounds from Curculigo orchioides

Six phenolic compounds isolated from Curculigo orchioides, including 2,6-dimethoxy benzoic acid (1), curculigoside A (2), curculigoside B (3), curculigine A (4), curculigine D (5) and 3,3′,5,5′-tetramethoxy-7,9′:7′,9-diepoxylignan-4,4′-di-O-β-d-glucopyranoside (6), together with the ethanol extract of Curculigo orchioides were evaluated for their activity on osteoblasts in neonatal rat calvaria cultures and multinucleated osteoclasts derived from rat marrow cells so as to characterize the antiosteoporotic components of this plant and explore the relationship of chemical structure with antiosteoporotic activity. The proliferation of osteoblast was assayed by MTT methods. The activity of ALP (alkaline phosphatase) and TRAP (tartrate-resistant acid phosphatase) was measured by p-nitrophenyl sodium phosphate assay. The TRAP stain was used to identify osteoclast in morphology. The resorption pit area on the bone slices formed by osteoclast was measured by computer image processing. The ethanol extract exhibited stimulatory effect on both the osteoblast proliferation and the ALP activity. Six compounds all increased the osteoblast proliferation, and compounds (1), (2) and (4) also slightly increased the osteoblastic ALP activity. Compounds (1), (2), (3), (6) and the ethanol extract decreased area of bone resorption pit, osteoclastic formation and TRAP activity. These results indicated that phenolic compounds are antiosteoporotic chemical constituents from Curculigo orchioides, and their activities are related with chemical structures.     

Osteoblast tissue-nonspecific alkaline phosphatase antagonizes and regulates PC-1

Tissue-nonspecific alkaline phosphatase (TNAP) is essential for bone matrix mineralization, but the central mechanism for TNAP action remains undefined. We observed that ATP-dependent (45)Ca precipitation was decreased in calvarial osteoblast matrix vesicle (MV) fractions from TNAP-/- mice, a model of infantile hypophosphatasia. Because TNAP hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP(i)), we assessed phosphodiesterase nucleotide pyrophosphatase (PDNP/NTPPPH) activity, which hydrolyzes ATP to generate PP(i). Plasma cell membrane glycoprotein-1 (PC-1), but not the isozyme B10 (also called PDNP3) colocalized with TNAP in osteoblast MV fractions and pericellular matrix. PC-1 but not B10 increased MV fraction PP(i) and inhibited (45)Ca precipitation by MVs. TNAP directly antagonized inhibition by PC-1 of MV-mediated (45)Ca precipitation. Furthermore, the PP(i) content of MV fractions was greater in cultured TNAP-/- than TNAP+/+ calvarial osteoblasts. Paradoxically, transfection with wild-type TNAP significantly increased osteoblast MV fraction NTPPPH. Specific activity of NTPPPH also was twofold greater in MV fractions of osteoblasts from TNAP+/+ mice relative to TNAP-/- mice. Thus TNAP attenuates PC-1/NTPPPH-induced PP(i) generation that would otherwise inhibit MV-mediated mineralization. TNAP also paradoxically regulates PC-1 expression and NTPPPH activity in osteoblasts.     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

TNF-α and IL-1β inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells

AimsJoint inflammation leads to bone erosion in rheumatoid arthritis (RA), whereas it induces new bone formation in spondyloarthropathies (SpAs). Our aims were to clarify the effects of tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) on osteoblast differentiation and mineralization in human mesenchymal stem cells (MSCs).Main methodsIn MSCs, expression of osteoblast markers was assessed by real-time PCR and ELISA. Activity of tissue-nonspecific alkaline phosphatase (TNAP) and mineralization were determined by the method of Lowry and alizarin red staining respectively. Involvement of RUNX2 in cytokine effects was investigated in osteoblast-like cells transfected with a dominant negative construct.Key findingsTNF-α (from 0.1 to 10 ng/ml) and IL-1β (from 0.1 to 1 ng/ml) stimulated TNAP activity and mineralization in MSCs. Addition of 50 ng/ml of IL-1 receptor antagonist in TNF-α-treated cultures did not reverse TNF-α effects, indicating that IL-1 was not involved in TNF-α-stimulated TNAP activity. Both TNF-α and IL-1β decreased RUNX2 expression and osteocalcin secretion, suggesting that RUNX2 was not involved in mineralization. This hypothesis was confirmed in osteoblast-like cells expressing a dominant negative RUNX2, in which TNAP expression and activity were not reduced. Finally, since mineralization may merely rely on increased TNAP activity in a collagen-rich tissue, we investigated cytokine effects on collagen expression, and observed that cytokines decreased collagen expression in osteoblasts from MSC cultures.SignificanceThe different effects of cytokines on TNAP activity and collagen expression may therefore help explain why inflammation decreases bone formation in RA whereas it induces ectopic ossification from collagen-rich entheses during SpAs.