Effects of 2-carba-cyclic phosphatidic acid derivatives on IL-1ß-stimulated human chondrocytes.

Osteoarthritis (OA) is a common joint disease characterized by the breakdown of subchondral bone and cartilage damage, most often affecting middle-aged and elderly people. Although the etiology of OA is still unknown, some reports suggest that inflammatory factors such as interleukin (IL)- 1ß mediate the progression of OA. To investigate the effect of IL-1ß and the possibility of treatment for OA, we applied 2-carba-cyclic phosphatidic acid (2ccPA) and its derivatives on human chondrocytes. 2ccPA is a synthesized phospholipid derived from a bioactive phospholipid mediator: cyclic phosphatidic acid (cPA). It has been previously reported that 2ccPA exhibits anti-inflammatory and chondroprotective effects in an OA animal model. 2ccPA and its ring-opened body (ROB) derivative significantly suppressed IL-1ß-induced upregulation of IL-6, matrix metalloproteinase-13, and cyclooxygenase-2, as well as the degradation of type II collagen and aggrecan. However, the other two derivatives, namely the deacylated and ring-opened deacylated bodies, showed little effect on an IL-1ß-exposed human chondrosarcoma cell-line. These data suggest that the intactness of 2ccPA and ROB is essential for anti-inflammatory effects on OA. Collectively, this study provides evidence that 2ccPA and ROB would be novel therapeutic agents for OA.

A Review of Cyclic Phosphatidic Acid and Other Potential Therapeutic Targets for Treating Osteoarthritis.

Osteoarthritis (OA), a chronic degenerative joint disease, is the most common form of arthritis. OA occurs when the protective cartilage that cushions the ends of bones gradually breaks down. This leads to the rubbing of bones against each other, resulting in pain and stiffness. Cyclic phosphatidic acid (cPA) shows promise as a treatment for OA. In this article, we review the most recent findings regarding the biological functions of cPA signaling in mammalian systems, specifically in relation to OA. cPA is a naturally occurring phospholipid mediator with unique cyclic phosphate rings at the sn-2 and sn-3 positions in the glycerol backbone. cPA promotes various responses, including cell proliferation, migration, and survival. cPA possesses physiological activities that are distinct from those elicited by lysophosphatidic acid; however, its biochemical origin has rarely been studied. Although there is currently no cure for OA, advances in medical research may lead to new therapies or strategies in the future, and cPA has potential therapeutic applications.

ELISA system for human endothelial lipase.

BACKGROUND: Endothelial lipase (EL) regulates the metabolism of HDL cholesterol (HDL-C). However, the role of EL in regulating plasma HDL-C concentrations and EL's potential involvement in atherosclerosis in humans has not been fully investigated due to the lack of reliable assays for EL mass. We developed an ELISA system for serum EL mass. METHODS: Human recombinant EL proteins, purified from cultured media of human EL-transfected Chinese hamster ovary cells, were used as antigen and calibrator. Two specific monoclonal antibodies were generated in mice against recombinant EL protein for a sandwich ELISA. We measured EL mass in human serum using EL recombinant protein as a calibration standard. RESULTS: The EL antibodies did not cross-react with lipoprotein lipase and hepatic triglyceride lipase. The detection limit of the ELISA was 20 pg/mL, which is approximately 10 times lower than that of previous ELISA systems. Recovery of spiked EL in serum was 90%-105%. Assay linearity was intact with a >4-fold dilution of serum. Intra- and interassay CVs were <5%. The serum EL mass in 645 human subjects was [mean (SE)] 344.4 (7.7) pg/mL (range 55.2-1387.7 pg/mL). Interestingly, serum EL mass was increased in patients with diagnosed cardiovascular disease and inversely correlated with serum HDL-C concentrations. There was no difference in EL mass between pre- and postheparin plasma samples. CONCLUSIONS: This ELISA should be useful for clarifying the impact of EL on HDL metabolism and EL's potential role in atherosclerosis.

Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method.

Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) from the hyperthermophile Thermotoga maritima was biochemically characterized with the aim of establishing a colorimetric assay for inorganic pyrophosphate (PPi). When heterologously expressed in Escherichia coli, T. maritima PPDK (TmPPDK) was far more stable any other PPDK reported so far: it retained >90% of its activity after incubation for 1 h at 80°C, and >80% of its activity after incubation for 20 min at pHs ranging from 6.5 to 10.5 (50°C). In contrast to PPDKs from protozoa and plants, this TmPPDK showed very long-term stability at low temperature: full activity was retained even after storage for at least 2 years at 4°C. TmPPDK was successfully applied to a novel colorimetric PPi assay, which employed (i) a PPi cycling reaction using TmPPDK and nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) from Saccharomyces cerevisiae and (ii) a NAD cycling reaction to accumulate reduced nitroblue tetrazolium (diformazan). This enabled detection of 0.2 µM PPi, making this method applicable for preliminary measurement of PPi levels in PCR products in an automatic clinical analyzer.

Structure of D-3-hydroxybutyrate dehydrogenase prepared in the presence of the substrate D-3-hydroxybutyrate and NAD+.

D-3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis catalyzes the reversible conversion between D-3-hydroxybutyrate and acetoacetate. The enzyme was crystallized in the presence of the substrate D-3-hydroxybutyrate and the cofactor NAD(+) at the optimum pH for the catalytic reaction. The structure, which was solved by X-ray crystallography, is isomorphous to that of the complex with the substrate analogue acetate. The product as well as the substrate molecule are accommodated well in the catalytic site. Their binding geometries suggest that the reversible reactions occur by shuttle movements of a hydrogen negative ion from the C3 atom of the substrate to the C4 atom of NAD(+) and from the C4 atom of NADH to the C3 atom of the product. The reaction might be further coupled to the withdrawal of a proton from the hydroxyl group of the substrate by the ionized Tyr155 residue. These structural features strongly support the previously proposed reaction mechanism of D-3-hydroxybutyrate dehydrogenase, which was based on the acetate-bound complex structure.

Enzymatic characterization of an amine oxidase from Arthrobacter sp. used to measure phosphatidylethanolamine.

Ethanolamine oxidase was screened with the aim of using it to establish a novel enzymatic phosphatidylethanolamine assay. Ethanolamine oxidase activity was detected in the crude extract of Arthrobacter sp., and the enzyme was purified more than 15-fold in three steps with a 54% yield. SDS-PAGE revealed the presence of only one band, which migrated, with an apparent molecular mass of 70 kDa. Biochemical characterization of the enzyme showed phenylethylamine to be the preferred substrate, with the highest kcat/Km value. The primary structure, determined by sequencing the cloned gene, showed a high degree of identity to Cu-containing phenylethylamine oxidase (64%). When heterologously overexpressed in Escherichia coli, the enzyme exhibited only a trace of amine oxidase activity, but high levels of activity emerged after exposure to Cu2+, as is typical of recombinant copper amine oxidases. Preliminary application of this enzyme coupled with phospholipase D for determination of phosphatidylethanolamine is also described. This is the first enzymatic method for the measurement of phosphatidylethanolamine.

An automated method for measuring lipoprotein lipase and hepatic triglyceride lipase activities in post-heparin plasma.

BACKGROUND: Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) play a central role in triglyceride-rich lipoprotein metabolism by catalyzing the hydrolysis of triglycerides. Quantification of LPL and HTGL activity is useful for diagnosing lipid disorders, but there has been no automated method for measuring these lipase activities. METHODS: The automated kinetic colorimetric method was used for assaying LPL and HTGL activity in the post-heparin plasma using the natural long-chain fatty acid 2-diglyceride as a substrate. LPL activity was determined with apoCII and HTGL activity was determined without apoCII with 2 channel of auto-analyzer. RESULTS: The calibration curve for dilution tests of the LPL and HTGL activity assay ranged from 0.0 to 500 U/L. Within-run CV was obtained within a range of 5%. No interference was observed in the testing of specimens containing potentially interfering substances. The measurement range of LPL activity in the post-heparin plasma was 30-153 U/L, while HTGL activity was 135-431 U/L in normal controls. CONCLUSIONS: The L PL and HTGL activity assays are applicable to quantitating the LPL and HTGL activity in the post-heparin plasma. This assay is more convenient and faster than radiochemical assay and highly suitable for the detection of lipid disorders.

The structures of pyruvate oxidase from Aerococcus viridans with cofactors and with a reaction intermediate reveal the flexibility of the active-site tunnel for catalysis.

The crystal structures of pyruvate oxidase from Aerococcus viridans (AvPOX) complexed with flavin adenine dinucleotide (FAD), with FAD and thiamine diphosphate (ThDP) and with FAD and the 2-acetyl-ThDP intermediate (AcThDP) have been determined at 1.6, 1.8 and 1.9 A resolution, respectively. Each subunit of the homotetrameric AvPOX enzyme consists of three domains, as observed in other ThDP-dependent enzymes. FAD is bound within one subunit in the elongated conformation and with the flavin moiety being planar in the oxidized form, while ThDP is bound in a conserved V-conformation at the subunit-subunit interface. The structures reveal flexible regions in the active-site tunnel which may undergo conformational changes to allow the entrance of the substrates and the exit of the reaction products. Of particular interest is the role of Lys478, the side chain of which may be bent or extended depending on the stage of catalysis. The structures also provide insight into the routes for electron transfer to FAD and the involvement of active-site residues in the catalysis of pyruvate to its products.

Alteration of substrate specificity of fructosyl-amino acid oxidase from Ulocladium sp. JS-103.

We showed by random mutagenesis that one-amino-acid substitution at Arg94 in fructosyl-amino acid oxidase from Ulocladium sp. JS-103 enhanced substrate specificity toward fructosyl valine (FV), a model compound of hemoglobin A(1c). Kinetic analysis showed that the specificity of the R94W mutant enzyme toward FV was 14-fold that of the wild-type enzyme. The mutant enzyme obtained will be useful in developing an enzymatic measurement method for hemoglobin A(1c).

The majority of lipoprotein lipase in plasma is bound to remnant lipoproteins: A new definition of remnant lipoproteins.

BACKGROUND: Lipoprotein lipase (LPL) is a multifunctional protein and a key enzyme involved in the regulation of lipoprotein metabolism. We determined the lipoproteins to which LPL is bound in the pre-heparin and post-heparin plasma. METHODS: Tetrahydrolipstatin (THL), a potent inhibitor of serine lipases, was used to block the lipolytic activity of LPL, thereby preventing changes in the plasma lipoproteins due to ex vivo lipolysis. Gel filtration was performed to obtain the LPL elution profiles in plasma and the isolated remnant lipoproteins (RLP). RESULTS: When ex vivo lipolytic activity was inhibited by THL in the post-heparin plasma, majority of the LPL was found in the VLDL elution range, specifically in the RLP as inactive dimers. However, in the absence of THL, most of the LPL was found in the HDL elution range as active dimers. Furthermore, majority of the LPL in the pre-heparin plasma was found in the RLP as inactive form, with broadly diffused lipoprotein profiles in the presence and absence of THL. CONCLUSIONS: It is suggested that during lipolysis in vivo, the endothelial bound LPL dimers generates RLP, forming circulating RLP-LPL complexes in an inactive form that subsequently binds and initiates receptor-mediated catabolism.

A highly sensitive chemiluminescent reverse transcriptase assay for human immunodeficiency virus.

A simple and highly sensitive reverse transcriptase (RT) assay was developed by combining a previously reported non-radioisotopic RT assay with the use of a template-primer-immobilized microplate, an enzyme capture protocol, product digestion and a chemiluminescent substrate. The assay was able to detect directly the RT activity in serum samples, plasma and cell culture medium without the need for concentration and extraction of the enzyme. The assay was able to detect RT activity equivalent to 100 virions/ml of HIV-1. These results suggest that this highly sensitive chemiluminescent RT assay can be used not only for virological investigation but also for routine screening of biopharmaceuticals.

A novel colorimetric assay for the determination of lysophosphatidic acid in plasma using an enzymatic cycling method.

BACKGROUND: Several methods for measuring concentrations of lysophosphatidic acid (LPA), a lipid mediator, have been reported to date. However, these methods are not routinely used because most of them require specialized instrument and a complicated protocol. METHODS: We developed a novel LPA assay using enzymatic cycling. LPA in a sample is hydrolyzed with lysophospholipase to glycerol-3-phosphate, followed by enzymatic cycling using glycerol-3-phosphate oxidase and glycerol-3-phosphate dehydrogenase. Amplified concentrations of hydrogen peroxides, a product of the enzymatic cycling, are then colorimetrically measured. RESULTS: This method was specific for LPA, being insensitive to the presence of phosphatidic acid or lysophosphatidylcholine. The within-run and between-run CVs were 1.31-1.32% and 0.73-1.03%, respectively. The recoveries of exogenous LPA added to plasma were 100.3-101.6%. In males, LPA concentrations (mean+/-S.D.) of human serum and EDTA-plasma were 0.41+/-0.14 and 0.08+/-0.02 micromol/l, respectively. In females, they were 0.41+/-0.12 and 0.09+/-0.02 micromol/l, respectively. CONCLUSIONS: This novel colorimetric assay for determination of LPA using enzymatic cycling is simple and highly sensitive. It can be used with an automatic analyzer. It may also be useful for further studies of the biological functions of LPA as well as clinical applications in various disorders.

Study of glycated amino acid elimination reaction for an improved enzymatic glycated albumin measurement method.

BACKGROUND: In order to improve enzymatic glycated albumin measurement, we studied the endogenous glycated amino acid elimination reaction and the new bromocresolpurple (BCP) method for albumin measurement. METHODS: In the assay, endogenous glycated amino acids are first eliminated by oxidation by ketoamine oxidase. Second, glycated albumin is hydrolyzed to glycated amino acids by proteinase digestion, and glycated amino acids are oxidized to produce hydrogen peroxide, which is quantitatively measured. Third, albumin is measured by the new BCP method. Finally, glycated albumin value is calculated as the percentage of glycated albumin in total albumin. RESULTS: Glycated amino acid concentrations in prepared total parenteral nutrition products were increased in direct proportion to storage time and temperature. The glycated amino acid elimination reaction using ketoamine oxidase may be able to eliminate more than 15 mmol/l glycated amino acids. The glycated albumin values of samples calculated from the albumin concentrations using the new BCP method accorded with those calculated with the HPLC method. Fundamental performances (linearity, dilution test, analytical recovery, within-run and between-run CVs, interference study) of the present method were good. Detection of glycated albumin by the present method was significantly correlated with detection of glycated albumin by the high-performance liquid chromatography method (r(p) = 0.995). CONCLUSIONS: This new improved method is free of interference by endogenous glycated amino acids and is unaffected by albumin concentration, and enables more accurate analysis of glycated albumin.

An enzymatic method for the measurement of glycated albumin in biological samples.

BACKGROUND: In order to determine glycated albumin more easily and rapidly, we developed a new enzymatic method for glycated albumin in blood samples. METHODS: The method involves use of albumin-specific proteinase, ketoamine oxidase and serum albumin assay reagent. In the assay, glycated albumin is hydrolyzed to glycated amino acids by proteinase digestion, and ketoamine oxidase oxidizes the glycated amino acids to produce hydrogen peroxide, which is quantitatively measured. Glycated albumin is calculated as the percentage of glycated albumin in total albumin. RESULTS: The calibration curve for glycated albumin concentration was linear (r(p)=0.999) between 0.0 and 50.0 g/l and that for albumin concentration was linear (r(p)=0.999) between 0.0 and 60.0 g/l. The analytical recoveries of exogenous glycated albumin added to serum were 100-102.5%. The within-run and between-run CVs were 0.45-0.67% and 1.09-1.26%, respectively. This method was free from interference by bilirubin, chyle, glucose, globulins and labile intermediate. Weak interference by hemoglobin and ascorbic acid was observed. Glycated albumin detected by the present method was significantly correlated with glycated albumin detected by high-performance liquid-chromatographic (HPLC) method (serum: r(s)=0.989, plasma: r(p)=0.992). CONCLUSIONS: This new enzymatic method is simple, rapid, allows multiple determinations and enables quantitative analysis of glycated albumin.

Determination of urinary myo-inositol concentration by an improved enzymatic cycling method using myo-inositol dehydrogenase from Flavobacterium sp.

BACKGROUND: To determine myo-inositol more accurately, we improved the enzymatic cycling method. METHODS: We screened myo-inositol dehydrogenase (MIDH; EC.1.1.1.18) from Flavobacterium sp., which was highly specific to myo-inositol. We measured urinary myo-inositol/creatinine ratio 2 h after 75-g oral glucose tolerance test (2 h MI) of 71 volunteers, and investigated the relationship between diabetes and urinary myo-inositol concentration. RESULTS: The calibration curve was linear (r = 1.00) up to 2000 micromol/l, and the detection limit was 10 micromol/l. Within-run and between-run CVs were 0.5-1.1% and 0.4-1.3%, respectively. The 2 h MI of impaired fasting glycemia (IFG; 65.1 +/- 46.6 mg/g Cr, P < 0.005), impaired glucose tolerance (IGT; 85.0 +/- 73.7 mg/g Cr, P < 0.001) and diabetes (163.4 +/- 73.7 mg/g Cr, P < 0.0001) increased significantly compared with that of normal glucose tolerance (NGT; 24.0 +/- 14.4 mg/g Cr). From receiver operating characteristic analyses on 2 h MI, with 50 mg/g Cr as a tentative cutoff value to detect diabetes, the sensitivity and specificity were 100% and 77%, respectively. With 40 mg/g Cr as a tentative cutoff value to detect NGT, the sensitivity and specificity were 74% and 85%, respectively. CONCLUSIONS: The myo-inositol measurement method demonstrated high specificity and yielded accurate results. The results of clinical trials suggested that 2 h MI could not only determine diabetes but also distinguish IFG and IGT from NGT.

Plasma activity of endothelial lipase impacts high-density lipoprotein metabolism and coronary risk factors in humans.

AIM: Endothelial lipase (EL) is a determinant of plasma levels of high-density lipoprotein cholesterol (HDL-C). However, little is known about the impact of EL activity on plasma lipid profile. We aimed to establish a new method to evaluate EL-specific phospholipase activity in humans. METHODS: Plasma samples were obtained from 115 patients with coronary artery disease (CAD) and 154 patients without CAD. Plasma EL protein was immunoprecipitated using an anti-EL monoclonal antibody after plasma non-specific immunoglobulins were removed by incubation with ProteinA. The phospholipase activity of the immunoprecipitated samples was measured using a fluorogenic phospholipase substrate, Bis-BODIPY FL C11-PC. RESULTS: The EL-specific phospholipase assay revealed that plasma EL activity was inversely correlated with HDL-C levels (R = -0.3088, p＜0.0001). In addition, the EL activity was associated with cigarette smoking. Furthermore, EL activity in CAD patients was significantly higher than that in nonCAD patients. Concomitantly, the HDL-C level in CAD patients were significantly lower than that in non-CAD patients. CONCLUSION: We have established a method for human plasma EL-specific phospholipase activity by combination of EL immunoprecipitation and a fluorogenic phospholipid substrate. Plasma EL activity was associated with not only plasma HDL-C levels but also the risks for CAD.

A new enzyme-linked immunosorbent assay system for human hepatic triglyceride lipase.

BACKGROUND: The objective of this study was to establish a new sandwich based enzyme linked immunosorbent assay (ELISA) for measuring the protein mass of human hepatic triacylglyceride lipase (HTGL). METHOD: Two mouse monoclonal antibodies raised against human HTGL were used for the sandwich ELISA. The post-heparin plasma (PHP) samples obtained at a heparin dose of 50 unit/kg from 124 normolipidemic subjects were used for this ELISA. RESULTS: The dynamic assay range of the developed ELISA for the HTGL was from 0.47 to 30 ng/ml. The CV was <7% in both intra- and inter-assays, and it did not cross-react with lipoprotein lipase or endothelial lipase (EL). The HTGL concentration in PHP showed a strong correlation with HTGL activity [n=121, r=0.778, p<0.001]. There was a weak relation of HTGL concentration against high-density lipoprotein cholesterol (HDL-C) [n=123, r=-0.229, p=0.011] but no relations against total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), small dense LDL, remnant like particles cholesterol (RLP-C) and RLP-TG were confirmed. Interestingly, a weak but positive correlation between HTGL concentration and EL concentration was shown [n=122, p=0.013, r=0.224]. CONCLUSION: These results indicate that this new sandwich ELISA for measuring HTGL concentration in PHP can be applied in a daily clinical practice.

A novel nucleoside kinase from Burkholderia thailandensis: a member of the phosphofructokinase B-type family of enzymes.

The genome of the mesophilic Gram-negative bacterium Burkholderia thailandensis contains an open reading frame (i.e. the Bth_I1158 gene) that has been annotated as a putative ribokinase and PFK-B family member. Notably, although the deduced amino acid sequence of the gene showed only 29% similarity to the recently identified nucleoside kinase from hyperthermophilic archaea Methanocaldococcus jannaschii, 15 of 17 residues reportedly involved in the catalytic activity of M. jannaschii nucleoside kinase were conserved. The gene was cloned and functionally overexpressed in Rhodococcus erythropolis, and the purified enzyme was characterized biochemically. The substrate specificity of the enzyme was unusually broad for a bacterial PFK-B protein, and the specificity extended not only to purine and purine-analog nucleosides but also to uridine. Inosine was the most effective phosphoryl acceptor, with the highest k(cat)/K(m) value (80 s(-1).mm(-1)) being achieved when ATP served as the phosphoryl donor. By contrast, this enzyme exhibited no activity toward ribose, indicating that the recombinant enzyme was a nucleoside kinase rather than a ribokinase. To our knowledge, this is the first detailed analysis of a bacterial nucleoside kinase in the PFK-B family.

A novel enzymatic method for measuring mizoribine 5'-monophosphate levels in serum.

Inosine 5'-monophosphate dehydrogenases (IMPDH) from Oceanobacillus iheyensis and Bacillus subtilis were biochemically characterized with the aim of establishing a mizoribine 5'-monophosphate (MZR-P) assay. MZR-P is the active metabolite of mizoribine, which has been successfully used as an immunosuppressive agent. A sensitive method for measuring the MZR-P concentration in serum, based on its ability to inhibit IMPDH, was developed. The method was applied to an automatic clinical analyzer, enabling measurement of MZR-P levels in a set of serum samples. MZR-P at concentrations of 0.1-13.6 (microg/ml (0.3-40 microM) in the samples could be measured with 0.7% within-run coefficients of variation.