Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia.
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Hypophosphatasia is an inborn error of metabolism characterized by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) and skeletal disease due to impaired mineralization of cartilage and bone matrix. We investigated two independently generated TNSALP gene knock-out mouse strains as potential models for hypophosphatasia. Homozygous mice (-/-) had < 1% of wild-type plasma TNSALP activity; heterozygotes had the predicted mean of approximately 50%. Phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5'-phosphate are putative natural substrates for TNSALP and all were increased endogenously in the knock-out mice. Skeletal disease first appeared radiographically at approximately 10 days of age and featured worsening rachitic changes, osteopenia, and fracture. Histologic studies revealed developmental arrest of chondrocyte differentiation in epiphyses and in growth plates with diminished or absent hypertrophic zones. Progressive osteoidosis from defective skeletal matrix mineralization was noted but not associated with features of secondary hyperparathyroidism. Plasma and urine calcium and phosphate levels were unremarkable. Our findings demonstrate that TNSALP knock-out mice are a good model for the infantile form of hypophosphatasia and provide compelling evidence for an important role for TNSALP in postnatal development and mineralization of the murine skeleton. (+info)
Measurement of plasma and intracellular S-adenosylmethionine and S-adenosylhomocysteine utilizing coulometric electrochemical detection: alterations with plasma homocysteine and pyridoxal 5'-phosphate concentrations.
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BACKGROUND: The relative changes in plasma and intracellular concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) may be important predictors of cellular methylation potential and metabolic alterations associated with specific genetic polymorphisms and/or nutritional deficiencies. Because these metabolites are present in nanomolar concentrations in plasma, methods of detection generally require time-consuming precolumn processing or metabolite derivatization. METHODS: We used HPLC with coulometric electrochemical detection for the simultaneous measurement of SAM and SAH in 200 microL of plasma, 10(6) lymphocytes, or 10 mg of tissue. Filtered trichloroacetic acid extracts were injected directly into the HPLC system without additional processing and were eluted isocratically. RESULTS: The limits of detection were 200 fmol/L for SAM and 40 fmol/L SAH. In plasma extracts, the interassay CV was 3.4-5.5% and the intraassay CV was 2.8-5.6%. The analytical recoveries were 96.8% and 97.3% for SAM and SAH, respectively. In a cohort of healthy adult women with mean total homocysteine concentrations of 7.3 micromol/L, the mean plasma value was 156 nmol/L for SAM and 20 nmol/L for SAH. In women with increased homocysteine concentrations (mean, 12.1 micromol/L), plasma SAH, but not SAM, was increased (P <0.001), and plasma pyridoxal 5'-phosphate concentrations were reduced (P <0.001). Plasma SAM/SAH ratios were inversely correlated with homocysteine concentrations (r = 0.73; P <0.01), and the SAM/SAH ratio in plasma was directly correlated with the intracellular SAM/SAH ratio in lymphocytes (r = 0.70; P <0.01). CONCLUSIONS: Increased homocysteine in serum is associated with an increase in SAH and a decrease in the SAM/SAH ratio that could negatively affect cellular methylation potential. Accurate and sensitive detection of these essential metabolites in plasma and in specific tissues should provide new insights into the regulation of one-carbon metabolism under different nutritional and pathologic conditions. (+info)
Endogenous ATP released by electrical field stimulation causes contraction via P2x- and P2y-purinoceptors in the isolated tail artery of rats.
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Electrical field stimulation (EFS) caused contraction of isolated tail arteries of rats. The EFS-induced contraction showed frequency-dependence and was entirely abolished by the sodium channel blocker tetrodotoxin (1 x 10(-7) M). The EFS-induced (at 20 Hz) contraction was reduced by about 60% in the presence of phentolamine (1 x 10(-6) M). Therefore, later experiments were carried out in the presence of phentolamine. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (1 x 10(-8)-1 x 10(-6) M) and basilen blue E-3G (3 x 10(-5)-5 x 10(-5) M), P2-receptor antagonists, significantly inhibited the contraction evoked by EFS. In addition, PPADS significantly inhibited the contractions induced by ATP (1 x 10(-4) M) and a selective P2x-receptor agonist, alpha,beta-methylene ATP (1 x 10(-6) M). In contrast, basilen blue E-3G did not inhibit alpha,beta-methylene ATP-induced contraction. The ecto-ATPase activator apyrase (5 and 10 U/ml) significantly reduced the EFS-induced contractions. These findings suggest that endogenous ATP released by EFS causes contractions of rat tail artery via both the P2x-receptors and P2y-receptors. (+info)
Functional characterization and mechanism of action of recombinant human kynurenine 3-hydroxylase.
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The mitochondrial outer membrane enzyme kynurenine 3-hydroxylase (K3H) is an NADPH-dependent flavin mono-oxygenase involved in the tryptophan pathway, where it catalyzes the hydroxylation of kynurenine. K3H was transiently expressed in COS-1 cells as a glutathione S-transferase (GST) fusion protein, and the pure recombinant protein (rec-K3H) was obtained with a specific activity of about 2000 nmol.min-1.mg-1. Rec-K3H was shown to have an optimum pH at 7.5, to use NADPH more efficiently than NADH, and to contain one molecule of non-covalently bound FAD per molecule of enzyme. The mechanism of the rec-K3H-catalyzed reaction was investigated by overall initial-rate measurements, and a random mechanism in which combination of the enzyme with one substrate does not influence its affinity for the other is proposed. Further kinetic studies revealed that K3H activity was inhibited by both pyridoxal phosphate and Cl-, and that NADPH-catalyzed oxidation occurred even in the absence of kynurenine if 3-hydroxykynurenine was present, suggesting an uncoupling effect of 3-hydroxykynurenine with peroxide formation. This observation could be of clinical interest, as peroxide formation could explain the neurotoxicity of 3-hydroxykynurenine in vivo. (+info)
Evidence that rat hepatocytes co-express functional P2Y1 and P2Y2 receptors.
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Previous studies have indicated the expression of multiple P2Y receptors by rat hepatocytes although they have not been identified. Here we show by reverse transcriptase-polymerase chain reaction (RT - PCR) that rat hepatocytes express mRNA encoding all of the four cloned rat P2Y receptors (P2Y(1), P2Y(2), P2Y(4) and P2Y(6)). The effects of UTP have been examined on single aequorin-injected rat hepatocytes. The [Ca(2+)](i) transients induced by UTP were indistinguishable from those induced by ATP in the same cell. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both UTP- and ATP-induced [Ca(2+)](i) transients. UDP, an agonist at the P2Y(6) receptor, failed to induce transients in hepatocytes, indicating that functional P2Y(6) receptors coupled to increased [Ca(2+)](i) are not expressed. The transients evoked by ADP were more sensitive to inhibition by suramin than those induced by either ATP or UTP. Within an individual cell, the transients induced by ATP and UTP were inhibited by the same concentration of suramin. This sensitivity of ATP and UTP responses to suramin suggests action through P2Y(2) rather than P2Y(4) receptors. Co-application of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) caused a decrease in frequency and amplitude of transients induced by ADP. ATP- and UTP-induced transients also displayed a decrease in amplitude in response to addition of PPADS, but this was accompanied by an increase in frequency of transients. In conclusion the data presented here are consistent with the co-expression of P2Y(1) and P2Y(2) receptors by rat hepatocytes. (+info)
cDNA cloning, purification, and characterization of mouse liver selenocysteine lyase. Candidate for selenium delivery protein in selenoprotein synthesis.
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Selenocysteine lyase (SCL) (EC 4.4.1.16) is a pyridoxal 5'-phosphate-dependent enzyme that specifically catalyzes the decomposition of L-selenocysteine to L-alanine and elemental selenium. The enzyme was proposed to function as a selenium delivery protein to selenophosphate synthetase in selenoprotein biosynthesis (Lacourciere, G. M., and Stadtman, T. C. (1998) J. Biol. Chem. 273, 30921-30926). We purified SCL from pig liver and determined its partial amino acid sequences. Mouse cDNA clones encoding peptides resembling pig SCL were found in the expressed sequence tag data base, and their sequences were used as probes to isolate full-length mouse liver cDNA. The cDNA for mouse SCL (mSCL) was determined to be 2,172 base pairs in length, containing an open reading frame encoding a polypeptide chain of 432 amino acid residues (M(r) 47, 201). We also determined the sequence of the N-terminal region of putative human SCL. These enzymes were shown to be distantly related in primary structure to NifS, which catalyzes the desulfurization of L-cysteine to provide sulfur for iron-sulfur clusters. The recombinant mSCL overproduced in Escherichia coli was a homodimer with the subunit M(r) of 47,000. The enzyme was pyridoxal phosphate-dependent and highly specific to L-selenocysteine (the k(cat)/K(m) value for L-selenocysteine was about 4,200 times higher than that for L-cysteine). Reverse transcriptase-polymerase chain reaction and Western blot analyses revealed that mSCL is cytosolic and predominantly exists in the liver, kidney, and testis, where mouse selenophosphate synthetase is also abundant, supporting the view that mSCL functions in cooperation with selenophosphate synthetase in selenoprotein synthesis. This is the first report of the primary structure of mammalian SCL. (+info)
Effects of spinally administered P2X receptor agonists and antagonists on the responses of dorsal horn neurones recorded in normal, carrageenan-inflamed and neuropathic rats.
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1. The function and role of P2X receptors in the spinal transmission of nociception was investigated using the selective P2X receptor agonists, alpha,beta-methylene ATP (alpha,beta-me ATP) and beta, gamma-methylene-L-ATP (beta,gamma-me-L-ATP) and the P2X receptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-disulphonate (PPADS) and suramin. 2. Intrathecal administration of 5 and 50 microg of beta,gamma-me-L-ATP produced a significant facilitation of the C-fibre evoked response and a tendency towards increased excitability of the post-discharge, but not Abeta-fibre evoked response of dorsal horn neurones recorded in normal animals. Administration of similar doses of alpha,beta-me ATP did not produce an overall change in the response of the neuronal population. 3. Peripheral administration of 20 microg of these agonists into the paw of the rat evoked firing in the dorsal horn neurones. 4. Intrathecal administration of the antagonists, suramin (50 and 500 microg) and PPADS (5, 50 and 500 microg), to normal animals and to animals with a model of neuropathy induced by spinal nerve ligation did not alter the evoked neuronal responses. In contrast, intrathecal administration of 500 microg of suramin to animals 3 h after the induction of carrageenan inflammation produced a significant inhibition of the C-fibre evoked response of the neurones. Similar inhibitions were also seen following high doses of intrathecal PPADS, although this did not reach significance. 5. These results suggest that spinal P2X receptors may play a role in the modulation of spinal nociceptive transmission following the development of inflammation, but that these receptors play at most a minor role in spinal nociceptive processing in normal and neuropathic animals. (+info)
Functional characterization of the P2X(4) receptor orthologues.
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1. The aim of this study was to functionally characterize the recombinant mouse P2X(4) receptor and to compare its pharmacological properties with those of the human and rat orthologues. 2. Whole cell recordings were made from rafts of HEK-293 cells stably expressing recombinant mouse, rat or human P2X(4) receptors, using Cs-aspartate containing electrodes (3 - 8 MOmega) in a HEPES-buffered extracellular medium. 3. The agonist potency of ATP at the three species orthologues was similar, with mean EC(50) values of 2.3 microM, 1.4 microM and 5.5 microM, respectively. 4. Adenosine-5'-tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X(4) receptors (EC(50)=2.6 and 3.0 microM), but was significantly less potent at the rat orthologue (EC(50)=20.0 microM). alpha,beta-methylene adenosine-5'-triphosphate (alpha,beta-meATP) also acted as a partial agonist, producing 29% of the maximum response at the mouse P2X(4) and 24% at the human P2X(4) receptor. 5. In contrast to the other species orthologues, alpha,beta-meATP failed to elicit a significant agonist response at rat P2X(4) receptors, and was found to act as an antagonist, with an IC(50) of 4.6 microM, against 10 microM ATP. 6. Mouse P2X(4) receptors were found to be sensitive to the antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (IC(50)=10.5 microM), as were human P2X(4) receptors (IC(50)=9.6 microM). The rat receptor however, showed a low sensitivity to PPADS (IC(50)>100 microM). 7. All three orthologues were relatively suramin-insensitive (IC(50)>100 microM) and insensitive to 1-[N, O-Bis(5-isoquinoline sulphonyl)benzyl]-2-(4-phenylpiperazine)ethyl]-5-isoquinoline sulphonamide (KN-62; IC(50)>3 microM). 8. Our results suggest that the pharmacological properties of the mouse receptor are most similar to the human P2X(4) receptor, and differ markedly from the rat receptor. (+info)