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An intracellular uricase from Bacillus fastidious A.T.C.C. 26904 was characterized and evaluated for serum uric acid assay by a patented kinetic uricase method. The active uricase was 151 kDa by gel filtration through Sephadex G-200. Both SDS/PAGE and matrix-assisted laser-desorption ionization-time-of-flight MS resolved a single polypeptide with a molecular mass of approx. 36.0 kDa. The N-terminal sequence was AERTMFYGKGDV. The optimum pH for this uricase ranged from 9.0 to 10.5. At pH 9.2, the Km (Michaelis-Menten constant) was 204+/-14 micromol/l (n=8) and the Ki (inhibition constant) for xanthine was 41+/-7 micromol/l (n=5). By analysing the data monitored within 5 min at 0.03 unit/ml uricase, this kinetic uricase method gave linear response to uric acid in reaction solution from 1.3 to 60 micromol/l. Aside from other common errors, 30 micromol/l xanthine in the reaction solution caused no error in this kinetic uricase method, while it caused negative error in the indirect equilibrium method by peroxidase-coupled assay of H2O2. Uric acid in clinical sera by this kinetic uricase method (Ck) closely and positively correlated with that from the indirect equilibrium method (Ce) (Ck = 0.008+1.081 x Ce, r>0.990, n=99). However, Bland-Altman analysis suggested inconsistency between Ck and Ce. These results indicated that this kinetic uricase method using this uricase was reliable for serum uric acid assay with enhanced resistance to xanthine besides other common errors.

Background Hepatitis B virus covalently closed circular DNA (HBV cccDNA) is assembled by histones and non-histones into a chromatin-like cccDNA minichromosome in the nucleus. The cellular histone acetyltransferase GCN5, displaying succinyltransferase activity, is recruited onto cccDNA to modulate HBV transcription in cells. Clinically, IFN-α is able to repress cccDNA. However, the underlying mechanism of IFN-α in the depression of cccDNA mediated by GCN5 is poorly understood. Here, we explored the effect of IFN-α on GCN5-mediated succinylation in the epigenetic regulation of HBV cccDNA minichromosome. Results Succinylation modification of the cccDNA minichromosome has been observed in HBV-infected human liver-chimeric mice and HBV-expressing cell lines. Moreover, histone H3K79 succinylation by GCN5 was identified in the system. Interestingly, the mutant of histone H3K79 efficiently blocked the replication of HBV, and interference with GCN5 resulted in decreased levels of HBV DNA, HBsAg, and HBeAg in the supernatant from de novo HBV-infected HepaRG cells. Consistently, the levels of histone H3K79 succinylation were significantly elevated in the livers of HBV-infected human liver-chimeric mice. The knockdown or overexpression of GCN5 or the mutant of GCN5 could affect the binding of GCN5 to cccDNA or H3K79 succinylation, leading to a change in cccDNA transcription activity. In addition, Southern blot analysis validated that siGCN5 decreased the levels of cccDNA in the cells, suggesting that GCN5-mediated succinylation of histone H3K79 contributes to the epigenetic regulation of cccDNA minichromosome. Strikingly, IFN-α effectively depressed histone H3K79 succinylation in HBV cccDNA minichromosome in de novo HepG2-NTCP and HBV-infected HepaRG cells. Conclusions IFN-α epigenetically regulates the HBV cccDNA minichromosome by modulating GCN5-mediated succinylation of histone H3K79 to clear HBV cccDNA. Our findings provide new insights into the mechanism by which IFN-α modulate the epigenetic regulation of HBV cccDNA minichromosome.

A patented kinetic uricase method was evaluated for serum uric acid assay. Initial absorbance of the reaction mixture before uricase action (A 0 ) was obtained by correcting the absorbance at 293 nm measured before the addition of uricase solution, and background absorbance (A b ) was predicted by an integrated method. Uric acid concentration in reaction solution was calculated from ∆A, the difference between A 0 and A b , using the absorptivity preset for uric acid. This kinetic uricase method exhibited CV<4.3% and recovery of 100%. Lipids, bilirubin, hemoglobin, ascorbic acid, reduced glutathione and xanthine <0.32 mmol/L in serum had no significant effects. ∆A linearly responded to 1.2 to 37.5 µmol/L uric acid in reaction solution containing 15 µl serum. The slope of linear response was consistent with the absorptivity preset for uric acid while the intercept was consistent with that for serum alone. Uric acid concentrations in clinic sera by different uricase methods positively correlated to each other. By Bland-Altman analysis, this kinetic uricase method accorded with that by quantifying the total change of UV absorbance on the completion of uricase reaction. These results demonstrated that this kinetic uricase method is reliable for serum uric acid assay with enhanced resistance to both xanthine and other common errors, wider range of linear response and much lower cost.

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Selective oxidation of ethanol to acetaldehyde catalyzed by alcohol dehydrogenase (ADH) in the presence of nicotinamide adenine dinucleotide (NAD + ) is widely used for enzymatic assay of serum ethanol by quantifying the indicator product, reduced nicotinamide adenine dinucleotide (NADH). Among ADH methods in use, however, the classical kinetic method is sensitive to factors affecting ADH activity while the equilibrium method shows lower efficiency and narrower range of linear response.1,2 Recently, a new kinetic strategy was established for enzymatic analysis by predicting, instead of direct assay, the detector signal after the completion of enzyme reaction with an integrated method, i.e. fitting the integrated rate equation to an enzyme reaction curve. [3][4][5][6][7][8] This kinetic strategy shows resistance to most factors affecting enzyme activity and much wider range of linear response. Therefore, this kinetic strategy is anticipated to be useful for serum ethanol assay with ADH action.However, there are some problems associated with serum ethanol assay by this kinetic method. ADH is inhibited by its products NADH and acetaldehyde.9 Preliminary analysis found out that the ignorance of acetaldehyde inhibition on ADH resulted in ∼10% deviation between this kinetic method and the equilibrium method. This kinetic strategy worked for glutathione assay by using the integrated rate equation that took into account product inhibition on glutathione-S-transferase. 7,10 With the availability of instantaneous acetaldehyde concentration, the integrated rate equation for ADH reaction taking into account the inhibition from both products can be derived. Thus this kinetic method may still be feasible for serum ethanol assay by taking product inhibition on ADH into account. Moreover, this kinetic method requires reaction data of substrate consumption as high as possible for analysis by the integrated rate equation using the predictor variable of reaction time. [3][4][5][6][7][8] The reversibility of the ADH reaction makes only data of <35% substrate consumption available for analysis. Consequently, an acetaldehyde scavenger such as semicarbazide has to be used to remove acetaldehyde to get reaction data of >80% substrate consumption. But this strategy results in a loss of the stoichiometric relationship between NADH and acetaldehyde, and the unavailability of instantaneous acetaldehyde concentration prevents the derivation of the integrated rate equation with the predictor variable of reaction time for ADH reaction taking product inhibition into account. Theoretically, product inhibition can be incorporated into the integrated rate equation for ADH reaction by solving a group of differential rate equations including that for acetaldehyde reaction to semicarbazide, provided that both instantaneous concentration of semicarbazide and the secondorder reaction rate constant for its reaction to acetaldehyde are available. The initial concentration of semicarbazide in ADH reaction solution can be preset at a value >100-fold of th...

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