Mathematical modeling for bioprocess optimization of a protein drug, uricase, production by Aspergillus welwitschiae strain 1-4.

Microbial uricase is effective protein drug used to treat hyperuricemia and its complications, including chronic gout, also in prophylaxis and treatment of tumor lysis and organ transplants hyperuricemia. Uricase is commonly used as diagnostic reagent in clinical analysis for quantification of uric acid in blood and other biological fluids. Also, it can be used as an additive in formulations of hair coloring agents. A newly isolated strain, Aspergillus sp. 1-4, was able to produce extracellular uricase on a medium containing uric acid as inducer. Phylogenetic analysis based on ITS region sequence analysis and phenotypic characteristics showed that Aspergillus sp. strain 1-4 is closely related to Aspergillus welwitschiae and its nucleotide sequence was deposited in the GenBank database and assigned sequence accession number MG323529. Statistical screening using Plackett-Burman design with 20 runs was applied to screen fifteen factors for their significance on uricase production by Aspergillus welwitschiae. Results of statistical analysis indicated that incubation time has the most significant positive effect on uricase production followed by yeast extract and inoculum size with the highest effect values of 13.48, 5.26 and 4.75; respectively. The interaction effects and optimal levels of these factors were evaluated using central composite design. The maximum uricase production was achieved at incubation time (5 days), yeast extract (2 g/L) and inoculum size (4 mL/50 mL medium) are the optimum levels for maximum uricase production (60.03 U/mL). After optimization, uricase production increased by 3.02-folds as compared with that obtained from the unoptimized medium (19.87 U/mL).

Low serum uric acid levels in chronic insomnia patients: A case-control study.

Recent studies have demonstrated the presence of oxidative stress in insomnia patients. Uric acid (UA) is regarded as one of the most important antioxidants that may attenuate oxidative stress. The aim of our study was to investigate whether there is an alteration of serum UA levels in chronic insomnia patients. The association between sleep quality and serum UA in chronic insomnia patients was also investigated. We recruited 300 chronic insomnia patients and 300 age- and gender-matched normal controls. The uricase-PAP method was used to measure the concentration of UA both in patient and normal control subjects. The Pittsburgh Sleep Quality Index (PSQI) was used to assess the sleep quality of chronic insomniac participants. As a result, significantly lower serum UA levels were observed in patients with chronic insomnia when compared with normal control subjects (279.56±65.80 vs. 299.10±61.17µmol/L, t=-3.991, p<0.001). Low serum UA levels were correlated with high PSQI scores in multiple linear regression models (ß=-0.322, p<0.001). Our results suggested that low serum UA levels were associated with the presence and severity of chronic insomnia.

High-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins.

High-throughput estimation of specific activities of an enzyme and its mutants in a group (enzyme/mutants) in cell lysates via high-throughput assay of their activities and separate immunoturbidimetric assay (ITA) of their proteins was proposed. Pseudomonas aeruginosa arylsulfatase (PAAS) and Bacillus fastidious uricase (BFU) served as two models. ITA employed 0.75 mg of antisera against PAAS or BFU as the reference in 96-well microplates to measure the difference of extinction at 340 and 700 nm. According to the calibration curve, ITA quantified the reference from 0.40 to about 2.4 µg. The consistency among the abundance of enzyme/mutants through ITA of proteins in cell lysates prepared under the same conditions supported their consistent immunological reactivity to the antisera. Specific activities of PAAS/mutants or BFU/mutants in cell lysates through ITA of proteins showed excellent proportionality to those carefully determined after purification. Receiver-operating-characteristic (ROC) analysis of specific activities through ITA of proteins gave a higher area-under-curve than those for ROC analyses of other activity indices, which allowed the recognition of a PAAS/mutant of 50% higher activity after cell amplification in high-throughput mode. Therefore, ITA of enzyme/mutants as proteins is promising to estimate their specific activities in cell lysates in high-throughput mode for quantitative comparison.

Size-selective QD@MOF core-shell nanocomposites for the highly sensitive monitoring of oxidase activities.

In this work, we proposed a novel and facile method to monitor oxidase activities based on size-selective fluorescent quantum dot (QD)@metal-organic framework (MOF) core-shell nanocomposites (CSNCPs). The CSNCPs were synthesized from ZIF-8 and CdTe QDs in aqueous solution in 40min at room temperature with stirring. The prepared CdTe@ZIF-8 CSNCPs , which have excellent water dispersibility and stability, displays distinct fluorescence responses to hole scavengers of different molecular sizes (e.g., H2O2, substrate, and oxidase) due to the aperture limitation of the ZIF-8 shell. H2O2 can efficiently quench the fluorescence of CdTe@ZIF-8 CSNCPs over a linearity range of 1-100nM with a detection limit of 0.29nM, whereas large molecules such as substrate and oxidase have very little effect on its fluorescence. Therefore, the highly sensitive detection of oxidase activities was achieved by monitoring the fluorescence quenching of CdTe@ZIF-8 CSNCPs by H2O2 produced in the presence of substrate and oxidase, which is proportional to the oxidase activities. The linearity ranges of the uricase and glucose oxidase activity are 0.1-50U/L and 1-100U/L, respectively, and their detection limits are 0.024U/L and 0.26U/L, respectively. Therefore, the current QD@MOF CSNCPs based sensing system is a promising, widely applicable means of monitoring oxidase activities in biochemical research.

La ecografía en el diagnóstico de la hiperuricemia asintomática y la gota / Ultrasound in the diagnosis of asymptomatic hyperuricaemia and gout

El aumento de la concentración sérica de uratos (hiperuricemia, mayor o igual a 7,0mg/dL) ocasiona cristales que promueven inflamación y lesión articular. La ecografía puede poner de manifiesto estos depósitos de urato. Su presencia obliga a considerar que un paciente con hiperuricemia en realidad padece gota asintomática, y que un enfermo con gota sin tofos subcutáneos puede tener gota tofácea. La información que ofrece la ecografía (signo del «doble contorno» y de concreciones hiperecogénicas simulando nubes) posibilita una clasificación de la hiperuricemia y de la gota más precisas. Además, esta información puede dar lugar a modificaciones relevantes en cuanto al proceder diagnóstico y terapéutico en los enfermos con hiperuricemia y gota (AU)

The increase in serum urate concentrations (hyperuricaemia, ≥7.0mg/dL) creates crystals, which promote inflammation and joint lesions. Ultrasonography can reveal these urate deposits. The presence of crystals suggests that a patient with hyperuricaemia is actually experiencing asymptomatic gout, and that a patient with gout without subcutaneous tophi could experience tophaceous gout. The information offered by ultrasound (double contour sign and hyperechoic concretions mimicking clouds) enables a more specific classification of hyperuricaemia and gout. Additionally, this information can lead to relevant changes in terms of the diagnosis and therapeutic approach for patients with hyperuricaemia and gout (AU)

An amperometric uric acid biosensor based on chitosan-carbon nanotubes electrospun nanofiber on silver nanoparticles.

A novel amperometric uric acid biosensor was fabricated by immobilizing uricase on an electrospun nanocomposite of chitosan-carbon nanotubes nanofiber (Chi-CNTsNF) covering an electrodeposited layer of silver nanoparticles (AgNPs) on a gold electrode (uricase/Chi-CNTsNF/AgNPs/Au). The uric acid response was determined at an optimum applied potential of -0.35 V vs Ag/AgCl in a flow-injection system based on the change of the reduction current for dissolved oxygen during oxidation of uric acid by the immobilized uricase. The response was directly proportional to the uric acid concentration. Under the optimum conditions, the fabricated uric acid biosensor had a very wide linear range, 1.0-400 µmol L(-1), with a very low limit of detection of 1.0 µmol L(-1) (s/n = 3). The operational stability of the uricase/Chi-CNTsNF/AgNPs/Au biosensor (up to 205 injections) was excellent and the storage life was more than six weeks. A low Michaelis-Menten constant of 0.21 mmol L(-1) indicated that the immobilized uricase had high affinity for uric acid. The presence of potential common interfering substances, for example ascorbic acid, glucose, and lactic acid, had negligible effects on the performance of the biosensor. When used for analysis of uric acid in serum samples, the results agreed well with those obtained by use of the standard enzymatic colorimetric method (P > 0.05).

PEGylation enhancement of pH stability of uricase via inhibitive tetramer dissociation.

OBJECTIVES: Previously, PEGylated uricase was demonstrated to maintain catalytic activity at pH 5.8, the isoelectric point of uricase, where native uricase ceases to function. To find out whether PEGylation could enhance pH stability of uricase, the enzyme activity to pH curve was completely characterized. METHODS: Complete characterization of the enzyme activity to pH curve, indicating an inverted bell-shaped relationship not previously documented, is presented. PEGylation enhancement of uricase stability at a pH lower than that commonly found in the liver, can be explored by dynamic dissociation of uricase using ultrafiltration and size-exclusion chromatography. KEY FINDINGS: The results suggest the role of PEGylation in enhanced pH stability is via inhibition of subunit disintegration. The mechanism of this effect is characterized by the wrapping of PEG chains around uricase, providing a flexible shell preventing subunit disintegration. The presence of notable PEGylation-induced changes in uricase supports this mechanism and include improved enzyme-substrate affinity and elevated thermal stability. CONCLUSIONS: Characterization of PEGylated uricase provides a basis for the rational design of therapeutic PEGylated proteins.

Uricemia y síndrome metabólico en pacientes con hipertensión arterial / Uricemia and metabolic syndrome in patients with arterial hypertension

Objetivo. Los niveles de uricemia se han asociado con el síndrome metabólico (SM). Sin embargo, la relación entre estas 2 variables en pacientes con hipertensión arterial (HTA) esencial no ha sido estudiada. Pacientes y métodos. Estudio observacional, transversal, de 592 pacientes con HTA esencial. Para la definición de SM se emplearon >=3 criterios de la ATP-III. Se excluyeron a los pacientes con tratamiento hipouricemiante. Resultados. La prevalencia de SM fue del 52% (IC del 95%: 48-56%) y aumentó gradualmente a medida que se incrementaba la uricemia (uricemia: <=4,7mg/dl, 36%; uricemia >=6,8mg/dl, 70%; p<0,001). Los enfermos hipertensos con SM mostraron una uricemia media más elevada que los que no tenían esta comorbilidad (6,1±1,5mg/dl vs 5,4±1,3mg/dl; p<0,0001). La prevalencia de hiperuricemia (varones: >=7,0mg/dl; mujeres: >=6,0mg/dl) en los pacientes hipertensos que no recibían tratamiento diurético fue del 24,3% (en aquellos con SM, 40,5% frente a un 11,4% en los que no tenían SM; p<0,001). En el análisis multivariante los triglicéridos (OR: 1,008; IC del 95%: 1,004-1,012; p<0,001) y el índice de masa corporal (IMC) (OR: 1,118; IC del 95%: 1,059-1,181; p<0,001) fueron predictores independientes de la uricemia. Conclusiones. En los pacientes con HTA esencial, aproximadamente la mitad padecen SM y uno de cada 4 presenta hiperuricemia. El determinante más relevante del incremento de la concentración sérica de uratos es el aumento del IMC(AU)

Objective. Serum urate levels have been associated with metabolic syndrome (MS). However, the relationship between these two variables in patients with essential arterial hypertension has not been studied. Patients and methods. A Cross-sectional study in 592 patients with essential hypertension. The MS was defined according to the ATP-III criteria. We excluded patients with hypouricemic treatment. Results. The prevalence of MS was 52% (95% CI, 48-56%) and there was a graded increase with increasing serum urate (uricemia <=.7 mg/dl, 36%; uricemia >=6.8 mg/dl, 70%, P < 0.001). Hypertensive patients with MS showed a higher mean uricemia than those without this comorbidity (6.1 ± 1.5 mg/dl versus 5.4 ± 1.3 mg/dl, P <0.0001). The prevalence of hyperuricemia (men, > 7.0 mg/dL; women, >6.0 mg/dL) in hypertensive patients without diuretic treatment, was 24% (in those with MS 40% versus 11% without MS). In multivariate analysis, triglycerides (OR = 1.008, CI 95%: 1.004-1.012, P < 0.001) and body mass index (BMI) (OR = 1.118, CI 95%: 1.059-1.181, P < 0.001), were independent predictors of serum uric acid levels. Conclusions. In patients with essential hypertension, about half have MS and one out of four has hyperuricemia. The most important determinant of hyperuricemia is BMI(AU)

Influencia de la historia natural de la enfermedad en el diagnóstico previo en pacientes con gota / Influence of the natural history of disease on a previous diagnosis in patients with gout

El diagnóstico clínico de la gota puede ser preciso en formas clínicamente típicas. Sin embargo, en las formas clínicas crónicas o atípicas dicha fiabilidad parece no darse en la práctica. Se ha estudiado una cohorte de 348 pacientes con diagnóstico de gota por cristales de urato, remitidos con una valoración diagnóstica definida, de los que se recogieron datos sobre la severidad de la enfermedad en cuanto a la distribución articular y la presencia de tofos. Se analizaron los datos de precisión respecto al diagnóstico de derivación según los parámetros de severidad clínica y los facultativos que remitían a los pacientes. La mayor precisión diagnóstica se observó en las formas monoarticulares precedentes tanto en áreas de urgencias como en medicina familiar, pero no así en los remitidos por otras especialidades. La presencia de formas oligopoliarticulares redujo significativamente la precisión del diagnóstico de referencia en todos los grupos de facultativos. La presencia de tofos no favoreció la precisión diagnóstica. Las formas crónicas y severas de la gota son frecuentemente mal evaluadas desde el punto de vista clínico (AU)

The clinical diagnosis of gout can be quite precise in clinically typical forms. However, in chronic or atypical forms, such precision tends to be diminished in clinical practice. A cohort of 248 patients with a diagnosis of urate crystal arthropathy was studied, sent with a definite clinical evaluation, and data such as severity of the disease, joint distribution and the presence of tophi were gathered. Precision data was analyzed with respect to the referral diagnosis according to the severity parameters and the type of physician sending the patient. The best diagnostic precision was seen in the monoarticular forms that were sent both by the emergency room as well as by family physicians, but not in those sent by other specialists. The presence of oligoarticular forms reduced significantly the diagnostic precision in all of the specialties referring patients. The presence of tophi did not improve diagnostic precision.Chronic and severe forms of gout are frequently wrongly evaluated from the clinical standpoint (AU)

Immobilization of uricase within polystyrene using polymaleimidostyrene as a stabilizer and its application to uric acid sensor.

A novel amperometric uric acid (UA) sensor has been developed by coating the surface of a gold electrode with a polystyrene (PS) membrane formed by 30 microL of a 30 mg mL(-1) PS chloroform solution combined with 30 microL of a 5 mg mL(-1) polymaleimidostyrene (PMS) solution as a dispersant for enzyme, uricase; this membrane has been successfully employed as an immobilization support for uricase. In the PS membrane, PMS forms micelle-like structures containing uricase in an active state. This immobilized uricase membrane permits the permeation of oxygen, which is consumed by the uricase reaction. A good linear relationship is obtained over the concentration range of 5-105 microM. The concentration of uric acid was determined at a negative potential based on the decrease in the reduction current of oxygen and the interference of l-ascorbic acid can be completely eliminated.

Preliminary investigations on a new disposable potentiometric biosensor for uric acid.

In this paper, uricase, catalase, and electron mediator were coimmobilized on the surface of the tin oxide (SnO2)/indium tin oxide (ITO) glass, to develop a disposable potentiometric uric acid biosensor. The SnO2/ITO glass was employed as a pH sensor, fabricated by sputtering SnO2 thin films on the ITO glass. 3-Glycidyloxypropyltrimethoxysilane (GPTS) was utilized to immobilize uricase, catalase and the electron mediator (ferrocenecarboxylic acid, FcA) on the sensing window. The experimental results reveal that the optimal weight ratio of uricase, FcA to catalase (CAT) is 4:1:2. The sensor responds linearly between 2 mg/dl and 7 mg/dl at pH 7.5, in 20 mM of test solution, with a correlation coefficient of 0.99213. Accordingly, no significant interference was observed when interfering substances, glucose, urea and ascorbic acid, were added to the uric acid solution. Moreover, the recorded voltage was relatively constant during the first 28 days of measurement. Consequently, a potentiometric uric acid biosensor was realized with the advantages of low cost and simple fabrication.

Concentraciones séricas de uratos y excreción urinaria de ácido úrico en individuos con síndrome metabólico / Serum urate levels and urinary uric acid excretion in subjects with metabolic syndrome

Fundamento y objetivos: La hiperuricemia es una de las alteraciones características del síndrome metabólico (SM), aunque no incluida como criterio diagnóstico. No se conoce con exactitud el mecanismo que causa la elevación del urato sérico en el SM. Pacientes y método: Estudio transversal, sin intervención farmacológica, sobre 141 individuos (National Cholesterol Education Program Adult Treatment Panel III: 105 con SM y 36 sin SM). Se comparan los valores de urato sérico entre individuos con y sin SM, y se analiza si la elevación del urato sérico se asocia a infraexcreción renal o sobreproducción. Se determina la asociación del urato sérico a índice HOMA. Resultados: El grupo con SM presentó valores de urato sérico significativamente más elevados (media [DE] 5,6 [1,6] frente a 4,6 [1,7] mg/dl, p = 0,002), y menor excreción urinaria (aclaramiento de ácido úrico 3,60 [2,41] frente a 4,65 [3,04] ml/min/m2, p = 0,049; fracción excretada del ácido úrico filtrado 7,15 [4,72] frente a 9,81 [6,78]%, p = 0,045). Las variables asociadas con los valores de urato sérico fueron el sexo (media de urato sérico en varón 6,1 [1,6] frente a mujer 4,9 [1,6] mg/dl, p < 0,001), el alcohol (bebedores 6,1 [1,8] frente a no bebedores 5,2 [1,6] mg/dl, p < 0,01), y el SM (presente 5,6 [1,6] frente a ausente 4,6 [1,7] mg/dl, p < 0,002). En el análisis multivariante, sólo el sexo y el SM se asociaron independientemente con los valores de urato sérico. Conclusiones: El presente estudio muestra unos valores de urato sérico significativamente más elevados entre individuos con SM, relacionado a su vez con una infraexcreción renal de uratos. No se observó asociación significativa de los valores de urato sérico con el índice HOMA

Background and objectives: Hyperuricemia is considered a feature of the metabolic syndrome (MS) despite serum uric acid (SUA) is not considered a diagnostic criterion. The main physiopathological disturbance leading to the increased SUA is not completely understood. Patients and method: Descriptive study without drug intervention including 141 subjects (NCEP-ATPIII: 105 with MS and 36 without MS). Serum UA levels were compared in subjects with and without MS. The mechanism of the rise in SUA levels was assessed (overproduction or decreased renal excretion). The relation of SUA levels to the HOMA index was also evaluated. Results: Subjects with MS showed significantly higher SUA levels (5.6 [1.6] vs 4.6 [1.7] mg/dl, p = 0.002), and lower urinary UA excretion than subjects without MS (UA clearance 3.60 [2.41] vs 4.65 [3.04] ml/min/m2, p = 0.049; excreted fraction of filtered UA 7.15 [4.72] vs 9.81 [6.78%], p = 0.045). Sex (male 6.1 [1.6] vs female 4.9 [1.6] mg/dl, p < 0.001), alcohol intake (drinkers 6.1 [1.8] vs non-drinkers 5.2 [1.6] mg/dl, p < 0.01), and MS (present 5.6 [1.6] absent 4.6 [1.7] mg/dl, p < 0.002), were significantly associated with SUA. In the multiple regression analysis, sex and MS were independently associated with SUA. Conclusions: This study demonstrates significantly higher SUA levels in subjects with MS. A decreased urinary UA excretion, instead of urate overproduction, was the leading mechanism to explain high SUA. Serum UA levels were not associated with the HOMA index

Combined approach to the analysis of recombinant protein drugs using hollow-fiber flow field-flow fractionation, mass spectrometry, and chemiluminescence detection.

The impurities present in recombinant protein drugs produced by large-scale refolding processes can not only affect the product safety but also interact with the expressed protein. To relate the impurity profile to conformation and functionality of the protein drug, analytical methods able not to degrade the sample components should be preferred. In this work, an urate oxidase (uricase) drug from Aspergillus flavus expressed in Saccharomyces cerevisiae, and a reagent-grade uricase from Candida sphaerica expressed in Escherichia coli, are analyzed by combining hollow-fiber flow field-flow fractionation with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI/TOFMS) and with chemiluminescence enzyme activity assay. Preliminary detection and identification of sample impurities is performed by means of conventional methods such as RP HPLC with electrospray ionization quadrupole-TOF MS and MALDI/TOFMS with SDS PAGE and 2D SDS PAGE. Results show that the recombinant uricase samples obtained from different microorganisms have different impurities and different enzymatic activity and that different uricase oligomers are present in solution.

The comparison of the estimation of enzyme kinetic parameters by fitting reaction curve to the integrated Michaelis-Menten rate equations of different predictor variables.

The estimation of enzyme kinetic parameters by nonlinear fitting reaction curve to the integrated Michaelis-Menten rate equation ln(S(0)/S)+(S(0)-S)/K(m)=(V(m)/K(m))xt was investigated and compared to that by fitting to (S(0)-S)/t=V(m)-K(m)x[ln(S(0)/S)/t] (Atkins GL, Nimmo IA. The reliability of Michaelis-Menten constants and maximum velocities estimated by using the integrated Michaelis-Menten equation. Biochem J 1973;135:779-84) with uricase as the model. Uricase reaction curve was simulated with random absorbance error of 0.001 at 0.075 mmol/l uric acid. Experimental reaction curve was monitored by absorbance at 293 nm. For both CV and deviation <20% by simulation, K(m) from 5 to 100 micromol/l was estimated with Eq. (1) while K(m) from 5 to 50 micromol/l was estimated with Eq. (2). The background absorbance and the error in the lag time of steady-state reaction resulted in negative K(m) with Eq. (2), but did not affect K(m) estimated with Eq. (1). Both equations gave better estimation of V(m). The computation time and the goodness of fit with Eq. (1) were 40-fold greater than those with Eq. (2). By experimentation, Eq. (1) yielded K(m) consistent with the Lineweaver-Burk plot analysis, but Eq. (2) gave many negative parameters. Apparent K(m) by Eq. (1) linearly increased, while V(m) were constant, vs. xanthine concentrations, and the inhibition constant was consistent with the Lineweaver-Burk plot analysis. These results suggested that the integrated rate equation that uses the predictor variable of reaction time was reliable for the estimation of enzyme kinetic parameters and applicable for the characterization of enzyme inhibitors.

Hipertensión e hiperuricemia / Hypertension and hyperuricemia

La asociación entre hipertensión e hiperuricemia es conocida. Los sujetos hipertensos con hiperuricemia presentan mayor riesgo de eventos cardiovasculares. La fisiopatología de la hiperuricemia en el sujeto hipertenso puede ser múltiple. Por una parte, la menor excreción de uratos puede estar asociada al tratamiento diurético, a la lesión renal hipertensiva (nefroesclerosis) o a la insulinorresistencia que con frecuencia acompaña a la hipertensión arterial. Por otro lado, la disfunción endotelial, presente en la hipertensión arterial, puede ocasionar una hipoxia tisular con aumento de los sustratos para la enzima xantina oxidasa (XO), cuyo producto final es el ácido úrico. Las consecuencias de esta hiperuricemia pueden ser lesiones renales y/o articulares gotosas. Además, la aparición de hiperuricemia denota bien un deterioro de la función renal o bien una sobreactividad de XO. Esta última es una fuente de radicales libres que son causa a su vez de disfunción endotelial y de resistencia a la insulina. La presencia de hiperuricemia en un sujeto hipertenso debe alertarnos para extremar las precauciones en el control global de su riesgo cardiovascular. (AU)

Bioconversion of poultry wastes I-factors influencing the assay and productivity of crude uricase by three uricolytic filamentous fungi.

The optimum temperature for biomass yield and uricase production by uricolytic fungi, Aspergillus terreus. A. flavus and Trichoderma sp. was at 30 degrees C. The time required for maximum production of uricase and biomass yield was 4 days for two Aspergillus species and 6 days for Trichoderma sp. The optimum pH was at 6.4 for A. terreus and pH 6.6 for both A. flavus and Trichoderma sp. The maximum fungal biomass yield was achieved in medium supplemented with 4% poultry waste. The best carbon sources for the production of uricase and mycelia yield were glycerol, sucrose and maltose by A. terreus, A. flavus and Trichoderma sp., respectively. Uric acid was found to be the best nitrogen source for production and activity of uricase by the three tested fungi. The addition of some vitamins to the culture media increased the maximum biomass yield of all the isolates, but did not significantly increase uricase production.

Bioconversion of poultry wastes. I--Factors influencing the assay and productivity of crude uricase by three uricolytic filamentous fungi.

The optimum temperature for biomass yield and uricase production by uricolytic fungi, Aspergillus terreus, A. flavus and Trichoderma sp. was at 30 degrees C. The time required for maximum production of uricase and biomass yield was 4 days for two Aspergillus species and 6 days for Trichoderma sp. The optimum pH was at 6.4 for A. terreus and pH 6.6 for A. flavus and Trichoderma sp. The maximum fungal biomass yield was achieved in medium supplemented with 4% poultry waste. The best carbon sources for the production of uricase and mycelia yield were glycerol, sucrose and maltose by A. terreus, A. flavus and Trichoderma sp., respectively. Uric acid was found to be the best nitrogen source for production and activity of uricase by the three tested fungi. The addition of some vitamins to the culture media increased the maximum biomass yield of all the isolates, although no significantly increased uricase production was found.

Immunocytochemical localization of a urate oxidase immunoreactive protein in the plasma membranes and membranes of the secretory/endocytic compartments of digestive gland cells of the mussel Mytilus galloprovincialis.

The subcellular compartmentalization of urate oxidase (UOX) in the digestive glands of mussels, Mytilus galloprovincialis Lmk, was studied by means of immunoblotting and immunocytochemistry, using an antibody raised in rabbit against rat liver UOX. Western blot analysis of subcellular fractions revealed an immunoreactive polypeptide with a molecular weight similar to the corresponding mammalian hepatic protein. This crossreactive polypeptide of 32 kDa was particle-bound yet not peroxisome-associated. In paraffin sections the antiserum specifically labeled the plasma membrane of the digestive gland epithelial cells and discrete regions within the perinuclear and apical portions of the digestive tubules and duct cells. By electron microscopy gold particles representing antigenic sites were found on the microvilli and the lateral plasma membrane as well as the membranes of the secretory/ endocytic compartments, that is, the Golgi complex, secretory and some endocytic vesicle membranes. Since the peroxisomal UOX-antibody exhibits a comparable immunoreactivity towards a urate-transporter channel protein in rat kidney proximal tubules and has been used for its molecular cloning (Leal-Pinto et al., 1997, J. Biol. Chem. 272, 617-625), we suggest that the membrane protein identified in mussel digestive glands could represent a homologous urate-transporter protein.

Aggregate formation and degradation of overexpressed wild-type and mutant urate oxidase proteins. Quality control of organelle-destined proteins by the endoplasmic reticulum.

To analyze the cellular response caused by the overexpression of proteins in subcellular compartments, we constructed four expression clones encoding wild-type peroxisomal urate oxidase (UO), truncated UO lacking the peroxisomal targeting signal (UOdC), and chimeric UOs with a mitochondrial targeting signal (MTS) at the N-terminus of UOdC (MUOdC) or UO (MUO). After transfection, we examined COS-1 and HEK293 cells by immunofluorescence and immunoelectron microscopy, transmission electron microscopy, and pulse-chase experiments. The overexpressed UO and UOdC formed large electron-dense aggregates with no limiting membrane in both the cytoplasm and the nucleus. The UO aggregates exhibited the crystalloid structure quite similar to that of rat liver peroxisomal cores, whereas the UOdC aggregates formed a loose mass consisting of small dense substructures. The overexpressed MUOdC and MUO, on the other hand, formed other types of aggregates which were distributed in the cytoplasm. They consisted of tubular and circular membrane structures, which were morphologically confirmed to be derived from the endoplasmic reticulum (ER). No immunolabeling signals for MUOdC and MUO were present free in the cytoplasm and most of them were associated with membrane structures, suggesting that overexpressed UO containing the MTS attached to the ER membranes soon after synthesis and segregated from the cytosolic compartment. All the UO aggregates were stained for ubiquitin antigen. Pulse-chase experiments in combination with proteasome inhibitors suggested that proteasomes did not contribute to the degradation of these products.

Determination of 15N isotopic enrichment and concentrations of allantoin and uric acid in urine by gas chromatography/mass spectrometry.

A method for the determination of 15N enrichment and concentration of allantoin and uric acid simultaneously in urine using gas chromatography/mass spectrometry (GC/MS) is described. The urine samples contained [1,3-15N2] uric acid and its oxidation product allantoin. The uric acid and allantoin were isolated using an AG1-X8 (Cl-form) anion-exchange column and heated with a mixture containing 1:1 dimethylformamide and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA). The tert-butyldimethylsilyl (TBDMS) derivatives of allantoin and uric acid formed were injected into a gas chromatograph interfaced with a mass spectrometer operated under electron impact ionization conditions. Isotope ratio measurements were made from the abundance of the M-57 ions at m/z 398, 399 and 400 for allantoin and at m/z 567 and 569 for uric acid. 15N2 allantoin (99 at.%) was produced from [1,3-15N2] uric acid by treatment with uricase and used as a standard. Quantitation of allantoin and uric acid was based on isotopic dilution by spiking the urine sample with known quantities of 99 at.% [15N] uric acid and allantoin internal standards. The observed isotope ratio measurements from the prepared standards matched the theoretical values. Coefficients of variation in measurements of isotope ratio and concentration were 0.2 and 0.5%, respectively. The method was applied in a study to measure the urinary recovery of [1,3-15N2] uric acid continuously infused for 8-10 h into the blood of four sheep each on two occasions. Within 24 h, 65.9 +/- 9.1% of the tracer was excreted in the urine unchanged. Little was converted into allantoin (approximately 7% of the dose). The total recovery (5 days) of the infused tracer averaged 69.5 +/- 7.6% as uric acid and 76.8 +/- 9.3% as the sum of uric acid and allantoin. Uricase activities in plasma, liver and kidney of sheep were also measured using [1,3-15N2] uric acid as a substrate. Uricase activity was estimated to be 0.6 mU g-1 wet tissue in the liver and there appeared to be none in plasma and kidney. The low uricase activities in sheep tissues appeared to explain the limited conversion of the intravenously administered [15N] uric acid to allantoin but did not explain the large quantities of allantoin excreted in urine (8.96 +/- 0.86 and 1.36 +/- 0.25 mmol d-1 for allantoin and uric acid, respectively). The GC/MS method for the determination of 15N enrichment and concentration of allantoin and uric acid in urine is accurate and precise and provides a useful tool for studies on uric acid and allantoin metabolism.