Role of the mouse ank gene in control of tissue calcification and arthritis.
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Mutation at the mouse progressive ankylosis (ank) locus causes a generalized, progressive form of arthritis accompanied by mineral deposition, formation of bony outgrowths, and joint destruction. Here, we show that the ank locus encodes a multipass transmembrane protein (ANK) that is expressed in joints and other tissues and controls pyrophosphate levels in cultured cells. A highly conserved gene is present in humans and other vertebrates. These results identify ANK-mediated control of pyrophosphate levels as a possible mechanism regulating tissue calcification and susceptibility to arthritis in higher animals. (+info)
Mitochondrial oxidative phosphorylation is a downstream regulator of nitric oxide effects on chondrocyte matrix synthesis and mineralization.
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OBJECTIVE: Increased chondrocyte nitric oxide (NO) and peroxynitrite production appears to modulate decreased matrix synthesis and increased mineralization in osteoarthritis (OA). Because NO inhibits mitochondrial respiration, this study was undertaken to directly assess the potential role of chondrocyte mitochondrial oxidative phosphorylation (OXPHOS) in matrix synthesis and mineralization. METHODS: We studied cultured human articular chondrocytes and immortalized costal chondrocytes (TC28 cells). We also assessed the effects of antimycin A and oligomycin (inhibitors of mitochondrial complexes III and V, respectively) on chondrocyte mitochondrial respiration, ATP synthesis, and inorganic pyrophosphate (PPi) generation, and the mineralizing potential of released matrix vesicles (MV). RESULTS: Articular chondrocytes and TC28 cells respired at comparable rates. Peroxynitrite and NO donors markedly suppressed respiration and ATP generation in chondrocytes. Because NO exerts multiple effects on chondrocytes, we investigated the primary functions of mitochondrial respiration and OXPHOS. To do so, we identified minimally cytotoxic doses of antimycin and oligomycin, which both induced intracellular ATP depletion (by 50-80%), attenuated collagen and proteoglycan synthesis, and blocked transforming growth factor beta from increasing intracellular ATP and elaboration of PPi, a critical inhibitor of hydroxyapatite deposition. Antimycin and oligomycin also abrogated the ability of the ATP-hydrolyzing enzyme plasma cell membrane glycoprotein 1 (PC-1) to increase chondrocyte PPi generation. Finally, MV from cells treated with antimycin or oligomycin contained less PPi and precipitated >50% more 45Ca. CONCLUSION: Chondrocyte mitochondrial reserve, as NO-sensitive mitochondrial respiration-mediated ATP production, appears to support matrix synthesis and PPi elaboration and to regulate MV composition and mineralizing activity. NO-induced depression of chondrocyte respiration could modulate matrix loss and secondary cartilage mineralization in OA. (+info)
ATP release by mechanically loaded porcine chondrons in pellet culture.
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OBJECTIVE: To determine whether ATP is released from chondrocytes during mechanical stimulation and whether degradation of ATP generates inorganic pyrophosphate in chondron pellet cultures. METHODS: Chondron pellets were formed from 1.6 x 10(6) cells that had been enzymatically isolated from porcine articular cartilage. ATP was measured in media from cultures at rest and during fluid movement and cyclic compression. ATP hydrolysis was examined by high-performance liquid chromatography following the addition of gamma32P-ATP to resting cultures. RESULTS: Pellet cultures at rest maintained a steady-state concentration of 2-4 nM ATP in 2 ml of medium. The ATP concentration increased 5-12-fold with cyclic compression (7.5 and 15 kPa at 0.5 Hz), then decreased to preloading levels within 60 minutes despite continued loading. A subsequent increase in pressure stimulated a further increase in ATP release, suggesting that chondrocytes desensitize to load. Cell viability was similar for pellets at rest and up to 24 hours after compression. ATP released in response to mechanical stimulation was inhibited 50% by 0.5 mM octanol, suggesting a regulated mechanism for ATP release. Exogenous ATP was rapidly hydrolyzed to pyrophosphate in resting cultures. CONCLUSION: The occurrence of basal levels of extracellular ATP in the presence of pyrophosphohydrolase activity indicates that ATP was continuously released by chondrocytes at rest. Considering that chondrocytes express purinoceptors that respond to ATP, we suggest a role for ATP in extracellular signaling by chondrocytes in response to mechanical load. ATP released by chondrocytes in response to mechanical load is a likely source of pyrophosphate in calcium pyrophosphate dihydrate crystal deposition diseases. (+info)
Vacuolar proton pyrophosphatase activity and pyrophosphate (PPi) in Toxoplasma gondii as possible chemotherapeutic targets.
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The addition of PP(i) promoted the acidification of a subcellular compartment in cell homogenates of Toxoplasma gondii tachyzoites, implying the presence of a proton-translocating pyrophosphatase. The proton gradient was collapsed by addition of the K(+)/H(+) antiporter nigericin, and was also inhibited by addition of the PP(i) analogue aminomethylenediphosphonate (AMDP). Both proton transport and PP(i) hydrolysis were dependent upon K(+), but Na(+) caused partial inhibition of these activities. PP(i) hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, NaF and to the thiol reagent N-ethylmaleimide. This activity was unaffected by common inhibitors of phosphohydrolases, except that NaO(3)V (sodium orthovanadate) stimulated the activity by 87%. Immunofluorescence microscopy, using antisera raised against conserved peptide sequences of a plant vacuolar pyrophosphatase, suggested that the pyrophosphatase in T. gondii tachyzoites was located in the plasma membrane and intracellular vacuoles of the parasite. High-field (31)P-NMR spectroscopy showed that PP(i )was more abundant than ATP in tachyzoites. Bisphosphonates (PP(i) analogues), drugs that are used in the treatment of bone diseases, inhibited proton transport and PP(i) hydrolysis in tachyzoite homogenates, and also inhibited intracellular proliferation of tachyzoites in tissue culture cells. (+info)
Elicitation of suspension-cultured tomato cells triggers the formation of phosphatidic acid and diacylglycerol pyrophosphate.
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Phosphatidic acid (PA) and its phosphorylated derivative diacylglycerol pyrophosphate (DGPP) are lipid molecules that have been implicated in plant cell signaling. In this study we report the rapid but transient accumulation of PA and DGPP in suspension-cultured tomato (Lycopersicon esculentum) cells treated with the general elicitors, N,N',N",N"'-tetraacetylchitotetraose, xylanase, and the flagellin-derived peptide flg22. To determine whether PA originated from the activation of phospholipase D or from the phosphorylation of diacylglycerol (DAG) by DAG kinase, a strategy involving differential radiolabeling with [(32)P]orthophosphate was used. DAG kinase was found to be the dominant producer of PA that was subsequently metabolized to DGPP. A minor but significant role for phospholipase D could only be detected when xylanase was used as elicitor. Since PA formation was correlated with the high turnover of polyphosphoinositides, we hypothesize that elicitor treatment activates phospholipase C to produce DAG, which in turn acts as substrate for DAG kinase. The potential roles of PA and DGPP in plant defense signaling are discussed. (+info)
Determination of single-nucleotide polymorphisms by real-time pyrophosphate DNA sequencing.
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The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as biallelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popular markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibility to common diseases. We have addressed the issue of SNP genotype determination by investigating variations within the Renin-Angiotensin-Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection technology. The method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. The technical platform employed comprises a highly automated sequencing instrument that allows the analysis of 96 samples within 10 to 20 minutes. In addition to each studied polymorphic position, 5-10 downstream bases were sequenced for acquisition of reference signals. Evaluation of pyrogram data was accomplished by comparison of peak heights, which are proportional to the number of incorporated nucleotides. Analysis of the pyrograms that resulted from alternate allelic configurations for each addressed SNP revealed a highly discriminating pattern. Homozygous samples produced clear-cut single base peaks in the expected position, whereas heterozygous counterparts were characterized by distinct half-height peaks representing both allelic positions. Whenever any of the allelic bases of an SNP formed a homopolymer with adjacent bases, the nonallelic signal was added to those of the SNP. This feature did not, however, influence SNP readability. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing primers and allows the determination of several closely located SNPs in a single run. (+info)
Structural and functional consequences of removal of the interdomain disulfide bridge from the isolated C-lobe of ovotransferrin.
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The interdomain disulfide bond present in the C-lobe of all the transferrins was postulated to restrict the domain movement resulting in the slow rate of iron uptake and release. In the present study, the conformational stability and iron binding properties of a derivative of the isolated C-lobe of ovotransferrin in which the interdomain disulfide bond, Cys478-Cys671 was selectively reduced and alkylated with iodoacetamide were compared with the disulfide intact form at the endosomal pH of 5.6. Pyrophosphate and chloride mediated iron release kinetics showed no difference between the disulfide-intact and disulfide-reduced/alkylated forms; the two protein forms yielded similar observed rate constants showing an apparent hyperbolic dependency for anion concentrations. The conformational stability evaluated by unfolding and refolding experiments was greater for the disulfide-intact form than for the disulfide-reduced/alkylated form: the deltaG(D)H2O values at 30 degrees C obtained by using urea were 9.0+/-0.8 and 6.0+/-0.4 kJ/mol for the former and latter protein forms, respectively, and the corresponding values obtained by using guanidine hydrochloride were 6.2+/-0.9 and 4.3+/-0.5 kJ/mol. The dissociation constant of iron (kd) was almost the same for the two protein forms, and it varied only subtly with urea concentrations but increased markedly with GdnHCl concentrations. The nonidentical values of deltaG(D)H2O and kd for urea and GdnHCl can be attributed to the ionic nature of the later denaturant, in which chloride anion may influence the structure and iron uptake-release properties of the ovotransferrin C-lobe. Taken together, we conclude that the interdomain disulfide bond has no effect on the iron uptake and release function but significantly decreases the conformational stability in the C-lobe. (+info)
The role of histidine-114 of Ssulfolobus acidocaldarius geranylgeranyl diphosphate synthase in chain-length determination.
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Sulfolobus acidocaldarius geranylgeranyl diphosphate synthase yields (all-E)-C(20) prenyl diphosphate as a final product. The three-dimensional model of the enzyme suggested that removing two bulky residues at 77 and 114 would allow additional prenyl-chain elongation. To test this, we examined several mutants with substitutions at 77 and/or 114. As a result, the mutants, F77G, F77G and H114A, F77G and H114G, H114A, and H114G gave C(30), C(45), C(50), C(30) and C(40) as the main long product, respectively. These observations indicate that histidine-114 plays a crucial role in chain-length determination along with phenylalanine-77. (+info)