Preparation and characterization of uricase in uricase-catalase liposomes prepared using borate buffer / 南方医科大学学报

<p><b>OBJECTIVE</b>To characterize the property of uricase loaded in uricase-catalase liposomes (BUCLPs) prepared using borate buffer.</p><p><b>METHODS</b>BUCLPs were prepared using reverse-phase evaporation, and the physicochemical properties of uricase in the prepared BUCLPs were examined.</p><p><b>RESULTS</b>The optimal temperature of BUCLP and URI was 40 degrees celsius, their optimal pH values were 8.0 and 8.5, and their Michaelis-Menten constants were 14.207 µmol/L and 13.623 µmol/L, respectively. Fluorescence intensity of nanoliposome-loaded uricase-catalase that bound to FITC was higher than that of uricase-catalase binding directly with FITC; the fluorescence intensity of BUCLP was higher than that of free uricase-catalase at 280 nm.</p><p><b>CONCLUSION</b>Uricase activity is enhanced after loading in uricase and catalase liposomes.</p>

Isolation, partial purification and characterization of thermophilic uricase from Pseudomonas otitidis strain SN4

Aims: The present study aimed at isolating new source of uricase producers from Malaysian hot springs together with partial purification and characterization of thermophilic uricase from novel strain. Methodology and results: A bacteria strain, designated as SN4, was found to have the ability to degrade uric acid. 16S rRNA analysis identified SN4 as Pseudomonas otitidis. Uricase was then extracted from SN4 and purification was performed via ammonium sulphate precipitation. The effects of temperature, pH and metal ions on partially purified uricase were evaluated. Results showed that 70% ammonium sulphate concentration gave the highest uricase activity at 4.18 U/mL compared to other concentrations. The molecular weight of the partially purified uricase was 33 kilodalton (kDa). The optimum temperature for uricase was 45 °C and its activity was highest at pH 8.0. Calcium ions and copper ions enhanced uricase activity while cobalt ions reduced uricase activity. Conclusion, significance and impact of study: Isolation and investigation of uricase producers from new sources such as thermophiles would increase availability and thermal stability of the uricase that could be used for significant purposes such as in biochemical and clinical applications.

Cloning and characterization of a thermostable urate oxidase from Microbacterium sp. strain ZZJ4-1 / 生物工程学报

In order to characterize a thermostable urate oxidase (Uox) from Microbacterium sp. strain ZZJ4-1, we cloned its gene (uox). The open reading frame of uox contained 894 base pairs and encoded a protein with 297 amino acids. Alignment of gene sequences indicated there was no obvious identity with the most reported uox and that 72% identity was found with uox from Arthrobacter globiformis. We inserted the gene into the plasmid pET-15b to construct an expression vector pET-15b-uox and got it induced expression in Escherichia coli BL21 (DE3). After the purification of the recombinant Uox by the HisBind column, we studied some properties of it. It was composed of subunits with a molecular mass of about 35 kDa. The optimal temperature and pH was 30 degrees C and pH 7.5. It was stable below 65 degrees C and from pH 8.5 to 11.0. The Km value was 0.22 mmol/L with the uric acid as the substrate. Ag+, Zn2+, CU2+ and SDS could totally inhibit its activity while Tween 20, Tween 80 and Triton X-100 had a slight promotion effect. The thermal stability of this enzyme was the most excellent among the reported recombinant Uox. Based on this property, it would be very useful in the application.

Síndrome de lisis tumoral en pediatría / Tumoral lysis syndrome in pediatrics

Tumor lysis syndrome is a metabolic emergency resulting from the rapid and massive destruction of tumor cells, spontaneously or secondary to cytolytic therapy for cancer. This results in a huge imbalance of internal environment by releasing large amounts of intracellular contents into the interstitial and intravascular space, with serious clinical consequences or even death. The pediatric population is especially at risk of tumor lysis syndrome because it has a high rate of fast-growing tumors, such as those of hematologic origin. Proper recognition of the risk factors that can cause this syndrome, as well as specific prevention and treatment has substantially decreased complications and improved survival in these patients.

El síndrome de lisis tumoral es una emergencia metabólica derivada de la rápida y masiva destrucción de células tumorales en forma espontánea o secundaria a terapia citolítica del cáncer. Esta situación produce un enorme desequilibrio del medio interno al liberarse grandes cantidades de contenido intracelular al espacio intersticial e intravascular, con consecuencias clínicas serias e incluso mortales. La población pediátrica está especialmente expuesta a sufrir síndrome de lisis tumoral ya que presenta una tasa elevada de tumores de rápido crecimiento, como son los de orígenes hematológicos. El adecuado reconocimiento de los factores de riesgo que pueden causar este síndrome, así como su prevención y tratamiento específicos han disminuido sustancialmente las complicaciones y mejorado la sobrevida de estos pacientes.

Recent Progress in the Management of Gouty Arthritis and Hyperuricemia / 대한내과학회지

There is an increasing incidence of gout and hyperuricemia worldwide. It is because the population is getting older, their life style is sedentary, and they take protein-enriched food. Gout is one of the most common but best controllable chronic diseases of adult. There have been recent advances in the understanding of underlying mechanisms and treatment of gout and hyperuricemia. This article is aimed to provide the practical review of the currently recommended practice of care and also to introduce some recently approved drugs. The management concept of hyperuricemia is changing because not only the gout but also the hyperuricemia appear to be independent risk factors for hypertension, renal disease and cardiovascular disease. Gout causes a significant individual and social burden and loss of working force. Still hyperuricemia in the gout patient is often under-treated by the patients themselves and by the physicians also. Once the acute gout attack is controlled, patients should be followed with goal-oriented treatment of hyperuricemia and other risk factors. Allopurinol has remained as a first-line treatment for chronic hyperuricemia, but uricosuric agents may also be considered in some patients. These drugs have provided good control of the disease in most gout patients until now but the elderly patients with gout often carry co-medications, contra-indication to these drugs, and risk of adverse drug reaction. Febuxostat is a nonpurine xanthine-oxidase inhibitor. It is a new agent approved by the US FDA and Korean FDA for the treatment of hyperuricemia in patients with gout which may be used when allopurinol is not tolerated or contraindicated. Pegloticase is the PEGylated urate oxidase which is very potent and so recently approved by the US FDA for the gout refractory to conventional treatment.

Construction, expression, purification and characterization of mutant of Aspergillus flavus urate oxidase / 生物工程学报

We converted the TGC codon (307-309 bp) of Aspergillus flavus urate oxidase (UOX) gene to a GCC codon by using fusion PCR techniques to produce a C103A mutant. This gene was cloned into expression vector pET-42a (+) and then transformed into Escherichia coli BL21 (DE3). The mutant protein (UOX-Ala103) was expressed in soluble form at high levels after induction with IPTG The expressed rUOX-Ala103 accounted for about 45% of total bacterial proteins, rUOX-Ala103 of up to 98% purity was obtained after purified using hydrophobic interaction and anion exchange. Western blotting showed that the anti-UOX antibody specifically recognized rUOX-Ala103. The mutant protein showed a 60% increased in vitro biological activities compared with native protein, and performed a good activity of degrading the uric acid in vivo.

An update on the management of hyperuricemia and gout / 대한내과학회지

Gout is a common chronic inflammatory arthritis that can lead to significant disability. Gout is one of the few rheumatologic diseases that can be diagnosed with certainty and can be cured with appropriate therapy. Alcohol and dietary consumption are related to hyperuricemia and gout attacks. A moderate intake of purine-rich vegetables or protein is not related to an increased risk of gout. A weight-reducing, calorie-restricted diet with moderate carbohydrate restriction was beneficial for gout patients and reduced the serum uric acid and frequency of gout attacks, although these findings need to be confirmed. Clinicians should consider therapeutic options that do not increase the serum uric acid when treating associated conditions in gout patients. The acute gout attack can be treated appropriately with non-steroidal anti-inflammatory drugs, colchicine, or glucocorticoids. Hypouricemic treatment reduces the uric acid concentration by inhibiting its production (allopurinol) or enhancing its excretion (benzbromarone). Allopurinol is the agent used most commonly, but the recommended dose often fails to control the serum uric acid. Benzbromarone effectively reduces the serum uric acid, but possible hepatotoxicity should be monitored. Febuxostat, a new xanthine oxidase inhibitor, was recently approved by the Federal Drug Administration (FDA). PEGylated uricase, a potent parenteral hypouricemic agent, is under investigation for the treatment of gout.

Expression in Escherichia coli, purification and enzymatic properties of porcine urate oxidase / 生物工程学报

The aims of this research were to construct prokaryotic expression vector containing the gene of porcine urate oxidase (pUOX), optimize the conditions of the expression of pUOX in recombinant Escherichia coli BL21(DE3), and analyze the in vitro activity and the enzymological properties of pUOX. The pUOX gene was amplified by RT-PCR from the extracted total RNA of porcine liver, and was inserted into the prokaryotic expression vector pET30a(+) to construct a recombinant expression vector pET30a(+)/pUOX. We identified the recombinant vector by endonuclease digestion and sequence analysis. The pUOX gene was amplified and cloned into the vector pET30a(+) successfully. And then the recombinant vector was transformed into E. coli BL21(DE3). The expression of pUOX with a molecular of approximately 41 kD was induced by IPTG. We also optimized the expression conditions of the recombinant protein. The recombinant protein was mostly located in the cytoplasm and it was insoluble. After the inclusion body was solved in 8 mol/L urea and refolding in 2 mol/L urea, the recombinant protein was collected and purified by Ni2+-NTA column. This recombinant protein had a specific activity of 50.61 IU/mg and showed similar properties of optimum temperature and thermal stability, base on the enzymatic assay and analysis of enzymological properties. These results would help to analyze the in vivo activity by testing animal.

Gota en el anciano / No presenta

La gota es la artritis crónica más común en el anciano. Puede ser fácilmente diagnosticada por la presencia de cristales de urato monosódico al examen microscópico del líquido articular. Existen algunas diferencias clínicas entre la gota del adulto y la del anciano. El tratamiento es altamente efectivo pero puede ocasionar serias reacciones adversas si no se tiene en cuenta la comorbilidad de este grupo.

Gout is the most common chronic arthritis in older people that can lead to significant and severe disability. It can be easy diagnosed with the presence of crystals of urate monosodic in the articular fluid. The clinical stages of gout include asymptomatic hyperuricemia, intermittent gouty arthritis, intercritical period and chronic tophaceous gout. There are some differences necessary to recognize to avoid errors in the management. Treatment of acute gout involves the use of NSAIDs, colchicine, corticosteroids or corticotropin (adrenocorticotropic hormone). Profilactic treatment includes the use of allopurinol and uricosuric agents; but all of these drugs could cause serious reactions in the elderly.

Inmunopatogenia de la gota / Immunopathogenesis of gout

La gota es un tipo de artritis gatillada por la cristalización de ácido úrico dentro de las articulaciones. Múltiples factores están involucrados en su desarrollo, constituyendo actualmente la hiperuricemia una condición estrechamente relacionada con el síndrome metabólico. Los humanos carecen de una enzima que degrada el ácido úrico llamada uricasa, lo que les confiere niveles de urato más elevados que otras especies animales. Se cree que esto sería un mecanismo adaptativo que entrega protección contra diversas noxas, dadas sus propiedades antioxidantes. Los cristales de urato monosódico pueden directamente iniciar la cascada inflamatoria a través de la activación del complemento y de diversos tipos celulares. Esto genera a nivel intracelular una cascada de transducción de señales que activarán un complejo catalítico multiproteico llamado inflamasoma, el cual es clave en la activación de caspasas inflamatorias, con el consecuente clivaje proteolítico de la pro-ILl en ILl , que junto a otras citoquinas y mediadores tendrán un rol fundamental en la patogénesis de esta enfermedad. Avances en el conocimiento de esta patología han permitido identificar nuevos targets terapéuticos y desarrollar nuevas terapias que han mostrado resultados favorables en pacientes con fracaso a los tratamientos convencionales.

Gout is a type of arthritis triggered by the crystallization of uric acid in the joints. Multiple factors are involved in its development, hyperuricemia currently constitutes a condition which is closely related to the metabolic syndrome. Humans lack an enzyme that degrades uric acid called uricase, which gives us urate levels higher than other animal species. It is believed that this is an adaptive mechanism that confers protection against various noxa, given its antioxidant properties. Monosodium urate crystals can directly start the infiammatory cascade through the activation of the complement and of various cell types. This leads to a cascade of intracellular signal transduction that will activate a multiprotein catalytic complex called infiammasome, which is key in the activation of infiamatory caspases and the consequent proteolytic cleavaje of the pro-ILl in ILl, which together with other cytokines and mediators have a fundamental role in the pathogenesis of this disease. Advances in the understanding of this condition have allowed us to identify new therapeutic targets and develop new therapies that have shown favorable results in patients that do not respond to conventional treatments.

Avoiding dialysis in tumour lysis syndrome: is urate oxidase effective? - a case report and review of literature

<p><b>INTRODUCTION</b>Hyperuricaemia in tumour lysis syndrome (TLS) can cause acute renal failure (ARF), necessitating dialysis. Recombinant urate oxidase (rasburicase) converts uric acid to soluble allantoin, which is excreted easily.</p><p><b>CASE REPORT</b>An 8-year-old boy with stage 3 Burkitt's lymphoma, TLS was successfully treated with hyper-hydration, diuretics and rasburicase, without dialysis. This is the first paediatric case in Kandang Kerbau Women's & Children's Hospital (KKH) in which rasburicase was used. We review the literature on the effectiveness of urate oxidase in avoiding dialysis in TLS.</p><p><b>TREATMENT AND OUTCOME</b>Our patient developed rapidly rising serum uric acid (SUA) and progressive renal impairment. Hyper-hydration and rasburicase (0.2mg/kg) were administered. SUA rapidly decreased from 1308 to 437 mmol/L within 12 hours. Urate oxidase has shown better results than allopurinol. There was a need for dialysis in 0.4% to 1.7% of patients with haematological malignancies given rasburicase, compared to 20% in patients given allopurinol.</p><p><b>CONCLUSIONS</b>Rasburicase can reverse renal insufficiency. Though expensive, it may be cost-effective by lowering incidence of dialysis, shortening the duration of intensive care and hospitalisation, allowing early chemotherapy.</p>

Evaluation of a kinetic uricase method for serum uric acid assay by predicting background absorbance of uricase reaction solution with an integrated method / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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 micromol/L uric acid in reaction solution containing 15 microl 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.

Serum cystatin C: a new predictor market for preeclampsia

To evaluate serum level of cystatin C, as early predictor for renal dysfunction in pregnant women developing preeclampsia, and to compare it as a new marker for preeclampsia with the use of serum creatinine or urate for the same diagnostic purpose. One hundred fifteen pregnant women were followed from the 12[th] week of pregnancy to full term. From whom, we got only ten preeclamptic women, and randomly eighteen normal pregnant women as a control group. Serum creatinine, urate and cystatin C were measured. Serum cystatin C, creatinine and urate were significantly increasing progressively in the 20[th] 28[th] and 36[th] weeks samples compared to the 12[th] weeks samples of the preeclamptic group only. The area under curve [AUC] in ROC analysis of cystatin C [0.76,0.79, and 0.95] was significantly greater than that of urate [0.62,0.67 and 0.76] and creatinine [0.43,0.38and 0.63]. Serum cystatin C was an early predictive marker for renal dysfunction in pregnant women developing preeclampsia, better than urate and creatinine. It has a superior diagnostic accuracy for preeclampsia compared to that of serum urate or creatinine in the 36th week sample. Also it has a superior prognostic accuracy in the 20th and 28th week samples as well [comparing AUC in ROC curves]

Paracetamol-induced liver toxicity in rats: light and electron microscopy and ultrahistochemical study

Twenty-four male albino rats were used to evaluate the toxic effect of both single acute dose of paracetamol [500 mg/Kg body weight] and chronic dose [50 mg/Kg body weight] once a day for 20 days. Paracetamol [Pa] was injected intraperitonially according to animal body weight. The ultra-structural changes of peroxisomes guided by two marker enzymes were studied. Light microscopy showed marked hepatotoxicity due to paracetamol depending on dose and time. Electron microscopy showed different changes in peroxisomal shape and enzyme contents. The changes were more pronounced following the chronic dose of [Pa]. The study identified the possible mechanism with which liver cells can modulate cellular sensitivity to toxic effect of Pa. Oxidative stress was hypothesized to contribute to the initiation or progression of Pa-induced hepatic injury and showed that peroxisomes were highly dynamic important organelles numerous in the liver cells and that one of its main function is detoxification

Oxidação de urato e tirosina por peroxinitrito: implicações para o desenvolvimento de seqüestradores e biomarcadores de peroxinitrito / Oxidation of urate and tyrosine by peroxynitrite: implications to the development of scavenger and biomarkers of peroxynitrite

Peroxinitrito (ONOOî+ONOOH), o produto da rápida reação do óxido nítrico com o ânion radical superóxido, tem recebido muita atenção como possível mediador dos efeitos deletérios associados a uma superprodução de `ANTPOT. PONTO NO'. O peroxinitrito é um potente oxidante que é capaz de oxidar e nitrar várias biomoléculas por mecanismos que contribuimos para esclarecer no decorrer desta tese. Especificamente, estudamos a oxidação de urato e tirosina por peroxinitrito. Demonstramos que o urato é oxidado por peroxinitrito a alantoina, aloxana e ao radical aminocarbonila. Como a reação direta entre urato e peroxinitrito tem uma constante de velocidade relativamente baixa (k= 4,8 x 10² Mîû.sîû) em comparação com àquelas de outras biomoléculas, sugerimos que o urato é um potente sequestrador dos radicais derivados do peroxinitrito (`ANTPOT. PONTO N'`O IND. 2' e C`O IND. 3 POT. PONTO î' na maioria dos ambientes biológicos; a pH ácido, o radical `ANTPOT. PONTO OH' também pode se tornar relevante)...

Urate oxidase electrode based on dissolved oxygen probe for urine uric acid determination.

A biosensor for the specific determination of uric acid in urine was developed using urate oxidase (EC 1.7.3.3) in combination with a dissolved oxygen probe. Urate oxidase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated teflon membrane to serve as enzyme electrode. The electrode response was maximum when 50 mM glycine buffer was used at pH 9.2 and 35 degrees C. The enzyme electrode response depends linearly on uric acid concentration between 5-40 microM with a response time of 5 min. The enzyme electrode is stable for more than 2 weeks and during this period over 35 assays were performed.

Replacement of larval by adult Malpighian tubules during metamorphosis of Melipona quadrifasciata anthidioides Lep. (Hymenoptera, Apidae, Meliponinae): morphological studies

In Melipona quadrifasciata anthidioides a stingless bee, the malpighian tubules (Mt) of the larvae are totally reabsorbed during pupation and replaced by others that will function in the adult. The dye exclusion test shows generalized celular death in the larvall tubules present in prepupae. The results obtained with the acid phosphatase reaction corroborate these findings. Between the end of larval phase and emergence of adult, the insect does not have functional malpighian tubules, but develops waste storaging cells, the urate cells, for nitrogenous waste inactivation. the morphology of degeneration of the larval and the arising of the adult Mt, as well as that of the urate cell on light microscopy are described. The apparent change in the connection point between intestine and malpighian tubules of larva and adult is also discussed.

Distribution and abundance of uricase-producing bacteria in soils

Thirty-six bacterial strains were isolated from 25 soil samples collected from several localities in Damietta. Soil composition had an effect on the magnitude of bacterial flora. The total count was higher in non-cultivated soils. The isolated species were tested for their uric acid degradation ability, then investigated for their uricolytic activity