Form Of Oxidase Research Articles

Comparative characteristics of blood xanthine oxidoreductase profiles in discoid and systemic lupus erythematosus

Discoid (DLE) and systemic lupus erythematosus (SLE) are chronic inflammatory diseases of the connective tissue. Along with pathogenetic features, they have common characteristics. Immune disorders and oxidative stress are involved in pathogenesis of DLE and SLE. Development of oxidative stress is closely associated with the activation of xanthine oxidoreductase (XOR) enzyme system.Aim of the study was to reveal the features of XOR activities profiles in blood of DLE and systemic lupus erythematosus (SLE) patients.Material and methods. 31 patients with DLE, 56 patients with SLE, 35 apparently healthy individuals were enrolled in study. The activities of two convertible forms of XOR (xanthine dehydrogenase (XDG; EC 1.17.1.4) and xanthine oxidase (XO; EC 1.17.3.2)) were measured in plasma, lysed WBC and lysed RBC. The level of uric acid (UA) was determined in plasma.Results and discussion. In contrast to the control, XO activity was higher and XDG activity was lower in plasma of DLE patients; activities of both XOR forms were decreased in lysed lymphocytes. Activity of XO was lower in plasma, XO and XDG activities were higher in lysed lymphocytes in DLE regardless of the SLE activity. XDG activity in plasma of and XO activity in lysed erythrocytes DLE patients was lower than in low activity SLE, but higher than in the subgroup with moderate and high disease activity. XDG activity of lysed erythrocytes in DLE was higher than in patients with low and moderate SLE activities, but lower than in patients with high diseases activity. Uric acid content in plasma of DLE patients was not differ from the control values, its level was increased at II and III degrees of SLE activity.Conclusions. Changes in XOR activity in plasma and lysed WBC in DLE and SLE are unidirectional, but are more pronounced in the systemic form of the disease. Thus, DLE and SLE are characterized by some common changes in XOR activity, at the same time, XOR blood profiles have distinctive features, which are characteristic for nosology.

A novel flavonol glycoside and six derivatives of quercetin and kaempferol from Clematis flammula with antioxidant and anticancer potentials

Clematis flammula leaves are traditionally used in Algeria to treat rheumatoid arthritis. Our aim was to identify the main compounds in this plant in order to characterize its antioxidant and anticancer activities.A new flavonol compound, kaempferol 3-O-[(6-O- caffeoyl)- glucosyl(1 → 2)]-(6-Ocaffeoyl) glucoside-7-O-rhamnoside (6) along with six known flavonol molecules were isolated from an ethanolic extract of Clematis flammula leaves. The chemical structures of these flavonols were elucidated using NMR and high resolution-MS spectroscopies. Antioxidant activities of the extract were revealed through its elimination of superoxide radical (O2.-) produced enzymatically (49.7 ± 1.52% at 50 μg/ml) and non-enzymatically (34 ± 1.2% at 100 μg/ml), probably related to its inhibition of the xanthine oxidase form of the xanthine oxidoreductase (XOR) enzyme (25.05 ± 2.33 μg/mL at 100 μg/mL), but mostly to that of the NADH oxidase form of the enzyme (69.16 ± 4.0%). Cytotoxicity tests of the extract on human hepatoma cell line HepG2 and ovarian cancer cell lines A2780 and OVCAR3 were promising especially regarding A2780 cell line (IC50: 77.0 μg/mL), which was comparable to taxol (IC50:76.9 μg/mL).

Indirect Antioxidant Effects of the Nitrite Anion: Focus on Xanthine Oxidase

One electron reduction of nitrite (NO2−) has been determined to be a significant, noncanonical source of nitric oxide (NO) with molybdopterin enzymes being identified as critical to this process. Of the molybdopterin enzymes identified as NO2− reductases, xanthine oxidoreductase (XOR) is the most extensively studied. Paradoxically, XOR generates oxidants and thus can contribute to oxidative stress under inflammatory conditions when the oxidase form (XO) of XOR is abundant. However, under similar inflammatory conditions XO has been associated with NO generation, especially when NO2− levels are elevated which begs the question: if reaction of nitrite with XO consumes electrons, then does it subsequently reduce oxidant generation? To address this question, electron paramagnetic resonance (EPR) was used, under controlled O2 tensions, to assess superoxide (O2·−) generation by endothelial-bound XO plus xanthine and the resultant impact of introducing NO2−. Nitrite diminished XO-derived O2·− under hypoxia (1% O2) whereas at 21% O2, it had no impact. To confirm these results and discount contributions from the reaction of NO with O2·−, molecular O2 consumption was assessed. The presence of NO2− decreased the rate of XO/xanthine-dependent O2 consumption in a concentration-dependent manner with greater impact under hypoxic conditions (1% O2) compared to 21% O2. In a more biologic setting, NO2− also diminished XO-dependent H2O2 formation in murine liver homogenates supplemented with xanthine. Interestingly, nitrate (NO3−) did not alter XO-dependent O2 consumption at either 21% or 1% O2; yet it did slightly impact nitrite-mediated effects when present at 2:1 ratio vs. NO2−. When combined, these data: 1) show a significant indirect antioxidant function for NO2− by decreasing oxidant generation from XO, 2) demonstrate that both XO-derived H2O2 and O2·− production are diminished by the presence of NO2− and 3) incentivize further exploration of the difference between XO reaction with NO2− vs. NO3−.

The mechanism and significance of the conversion of xanthine dehydrogenase to xanthine oxidase in mammalian secretory gland cells

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) occurs only in mammalian species. In fresh bovine milk, the enzyme exists predominantly as the oxidase form, in contrast to various normal organs where it is found primarily as the dehydrogenase: the mechanism of conversion to the oxidase in milk remains obscure. A systematic search for the essential factors for conversion from XDH to XO has been performed within fresh bovine milk using the highly purified dehydrogenase form after removal endogenous oxidase form by fractionation analysis. We find that conversion to the oxidase form requires four components under air: lactoperoxidase (LPO), XDH, SCN−, and substrate hypoxanthine or xanthine; the contribution of sulfhydryl oxidase appears to be minor. Disulfide bond formation between Cys-535 and Cys-995 is principally involved in the conversion, consistent with the result obtained from previous work with transgenic mice. In vitro reconstitution of LPO and SCN− results in synergistic conversion of the dehydrogenase to the oxidase the presence of xanthine, indicating the conversion is autocatalytic. Milk from an LPO knockout mouse contains a significantly greater proportion of the dehydrogenase form of the enzyme, although some oxidase form is also present, indicating that LPO contributes principally to the conversion, but that sulfhydryl oxidase (SO) may also be involved to a minor extent. All the components XDH/LPO/SCN− are necessary to inhibit bacterial growth in the presence of xanthine through disulfide bond formation in bacterial protein(s) required for replication, as part of an innate immunity system in mammals. Human GTEx Data suggest that mRNA of XDH and LPO are highly co-expressed in the salivary gland, mammary gland, mucosa of the airway and lung alveoli, and we have confirmed these human GTEx Data experimentally in mice. We discuss the possible roles of these components in the propagation of SARS-CoV-2 in these human cell types.

Comparative study of the biochemical properties of membrane-bound and soluble polyphenol oxidase from Prunus mume

Browning occurs during fruit and vegetable processing mainly as a result of oxidation by polyphenol oxidases. We compared the biochemical properties of membrane-bound polyphenol oxidase (mPPO) and soluble polyphenol oxidase (sPPO) isolated and purified from Prunus mume. The sPPO showed a greater affinity for the substrate catechol. The highest activity of sPPO was catechol, while those of the mPPO were catechol and 4-methylcatechol. The optimum pH of sPPO was 5.0, while that of the mPPO were 4.5 and 5.5; the optimum temperatures of sPPO and mPPO were 40 °C and 60 °C, respectively; and the sPPO was more resistant to acidic environments, while the mPPO maintained better stability within temperature changes. The metal ions Mg2+ and Na+ were able to activate both sPPO and mPPO of P. mume, while Al3+ and K+ significantly inhibited their activities, and both Cu2+ and Ca2+ inhibited the activity of sPPO but activated that of mPPO. Different inhibitors were found to have different effects on sPPO and mPPO, however, sodium sulfite, sodium bisulfite, l-cysteine, glutathione and ascorbic acid had obvious inhibitory effects on both forms of polyphenol oxidase. Provide theoretical support for the research on the characteristics and browning mechanism of polyphenol oxidase in fruits and vegetables.

Allopurinol blocks aortic aneurysm in a mouse model of Marfan syndrome via reducing aortic oxidative stress.

Increasing evidence indicates that redox stress participates in MFS aortopathy, though its mechanistic contribution is little known. We reported elevated reactive oxygen species (ROS) formation and NADPH oxidase NOX4 upregulation in MFS patients and mouse aortae. Here we address the contribution of xanthine oxidoreductase (XOR), which catabolizes purines into uric acid and ROS in MFS aortopathy. In aortic samples from MFS patients, XOR protein expression, revealed by immunohistochemistry, increased in both the tunicae intima and media of the dilated zone. In MFS mice (Fbn1C1041G/+), aortic XOR mRNA transcripts and enzymatic activity of the oxidase form (XO) were augmented in the aorta of 3-month-old mice but not in older animals. The administration of the XOR inhibitor allopurinol (ALO) halted the progression of aortic root aneurysm in MFS mice. ALO administrated before the onset of the aneurysm prevented its subsequent development. ALO also inhibited MFS-associated endothelial dysfunction as well as elastic fiber fragmentation, nuclear translocation of pNRF2 and increased 3'-nitrotyrosine levels, and collagen maturation remodeling, all occurring in the tunica media. ALO reduced the MFS-associated large aortic production of H2O2, and NOX4 and MMP2 transcriptional overexpression. Allopurinol interferes in aortic aneurysm progression acting as a potent antioxidant. This study strengthens the concept that redox stress is an important determinant of aortic aneurysm formation and progression in MFS and warrants the evaluation of ALO therapy in MFS patients.

Purification and comparison of soluble and membrane-bound polyphenol oxidase from potato (Solanum tuberosum) tubers

Enzymatic browning greatly affects the quality of potato products. Polyphenol oxidase (PPO) is the enzyme mainly responsible for potato enzymatic browning. PPO has soluble polyphenol oxidase (sPPO) and membrane-bound polyphenol oxidase (mPPO) forms. In this study, the properties of sPPO and mPPO were investigated in potato tubers. The molecular weight of potato sPPO and mPPO were estimated to be 69 kDa in the form of homodimers in vivo. The mass spectrometry results showed that the purified sPPO and mPPO protein in potato tubers was mainly tr|M1BMR6 (Uniprot). The optimum pH for sPPO and mPPO was 6.5, and the optimum temperatures were 20 and 30 °C, respectively. The Michaelis constant (Km) and maximum unit enzyme activity (Vmax) of sPPO were 6.08 mM and 2161 U/S when catechol was used as the substrate, whereas those of mPPO were 2.95 mM and 2129.53 U/S, respectively. The mPPO had stronger affinity to the substrate catechol than sPPO, whereas pyrogallic acid was stronger affinity for sPPO. Ascorbic acid and sodium sulfite were inhibitors of sPPO and mPPO, respectively. After understanding the different binding states of polyphenol oxidase, different inhibitors and treatment methods can be used to treat the enzyme according to different enzymatic properties, so as to achieve a greater degree of Browning control. These results will provide a theoretical basis for regulating PPO activity to reduce enzymatic browning during potato processing.

Recombinant Extracellular Cholesterol Oxidase from Nocardioides simplex.

Cholesterol oxidase is a highly demanded enzyme used in medicine, pharmacy, agriculture, chemistry, and biotechnology. It catalyzes oxidation of 3β-hydroxy-5-ene- to 3-keto-4-ene- steroids with the formation of hydrogen peroxide. Here, we expressed 6xHis-tagged mature form of the extracellular cholesterol oxidase (ChO) from the actinobacterium Nocardioides simplex VKM Ac-2033D (55.6 kDa) in Escherichia coli cells. The recombinant enzyme (ChONs) was purified using affinity chromatography. ChONs proved to be functional towards cholesterol, cholestanol, phytosterol, pregnenolone, and dehydroepiandrosterone. Its activity depended on the structure and length of the aliphatic side chain at C17 atom of the steroid nucleus and was lower with pregnenolone and dehydroepiandrosterone. The enzyme was active in a pH range of 5.25÷6.5 with the pH optimum at 6.0. Kinetic assays and storage stability tests demonstrated that the characteristics of ChONs were generally comparable with or superior to those of commercial ChO from Streptomyces hygroscopicus (ChOSh). The results contribute to the knowledge on microbial ChOs and evidence that ChO from N. simplex VKM Ac-2033D is a promising agent for further applications.

Interaction between PET tracer and the specific residues around the gate of the open form of Monoamine Oxidase B (MAO-B)

Monoamine Oxidase B (MAO-B) is the enzyme that metabolizes a monoamine neurotransmitter. Recently, a PET tracer targeting MAO-B, SMBT-1, has been developed as the biomarker of neurodegenerative diseases. However, the detailed binding mode of SMBT-1 has not been clear. To clarify the binding mode of SMBT-1 on MAO-B, we performed the molecular dynamics (MD) simulations of MAO-B in the outer mitochondrial membrane and the complex of MAO-B to SMBT-1, and the docking simulation of MAO-B with SMBT-1. We found that the Leu88, Leu171, Ile199, and Tyr326 around the substrate-binding site interact with SMBT-1. These hydrophobic residues mainly support the two aromatic rings of the center of SMBT-1, making the stable binding of SMBT-1.

Human Vascular Smooth Muscle Function and Oxidative Stress Induced by NADPH Oxidase with the Clinical Implications.

Among reactive oxygen species, superoxide mediates the critical vascular redox signaling, resulting in the regulation of the human cardiovascular system. The reduced form of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, NOX) is the source of superoxide and relates to the crucial intracellular pathology and physiology of vascular smooth muscle cells, including contraction, proliferation, apoptosis, and inflammatory response. Human vascular smooth muscle cells express NOX1, 2, 4, and 5 in physiological and pathological conditions, and those enzymes play roles in most cardiovascular disorders caused by hypertension, diabetes, inflammation, and arteriosclerosis. Various physiologically active substances, including angiotensin II, stimulate NOX via the cytosolic subunits’ translocation toward the vascular smooth muscle cell membrane. As we have shown, some pathological stimuli such as high glucose augment the enzymatic activity mediated by the phosphatidylinositol 3-kinase-Akt pathway, resulting in the membrane translocation of cytosolic subunits of NOXs. This review highlights and details the roles of human vascular smooth muscle NOXs in the pathophysiology and clinical aspects. The regulation of the enzyme expressed in the vascular smooth muscle cells may lead to the prevention and treatment of human cardiovascular diseases.

IDENTIFIKASI DAN UJI SENSITIVITAS BAKTERI Neisseria gonorrhoeae TERHADAP ANTIBIOTIK SEFIKSIM PADA PEKERJA SEKS KOMERSIAL DI PUSKESMAS II DENPASAR SELATAN

Gonorrhea is a sexually transmitted disease caused by Neisseria gonorrhoeae. Gonorrhea infection varies greatly in the community, especially in sexually active women. Infection cases in women are often asymptomatic, that can causes complication can easily occur. The aims of this study are to identify and determine sensitivity of Neisseria gonorrhoeae bacteria against cefixime antibiotics. This study was using a descriptive method and used 30 samples. Neisseria gonorrhoeae was isolatd from the vaginal swab sample of Commercial Sex Workers at Puskesmas II Denpasar Selatan. The cultured bacteria on Thayer Martin media were then identified by gram staining, oxidase test, and catalase test. Neisseria gonorrhoeae sensitivity test against cefixime was performed by disc diffusion method. The results of identification showed that 6 (20 %) of 30 samples were Neisseria gonorrhoeae bacteria with cell characteristics in the form of diplococcus, gram-negative, oxidase and catalase positive. The results of the sensitivity test showed that all isolats of Neisseria gonorrhoeae were resistant to cefixime

A new l-arginine oxidase engineered from l-glutamate oxidase.

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 catalyzes the oxidative deamination of l-glutamate to produce 2-ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l-glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l-glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l-arginine. The oxidative deamination activity of LGOX to l-arginine is higher than that of l-arginine oxidase form from Pseudomonas sp. TPU 7192. X-ray crystal structure analysis revealed that the guanidino group of l-arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild-type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l-arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l-arginine by LGOX R305E, is proportional to the concentration of l-arginine in a range from 0 to 100 μM. The linear relationship is maintained around 1 μM of l-arginine. Thus, LGOX R305E is suitable for the determination of l-arginine.

Xanthine Oxidoreductase Is Involved in Chondrocyte Mineralization and Expressed in Osteoarthritic Damaged Cartilage.

Pathologic calcification of cartilage consists of the formation of basic calcium phosphate (BCP) and/or calcium pyrophosphate dihydrate (CPPD) containing calcium crystals in mature hyaline or articular cartilage and is associated with aging, cartilage injury and likely plays a role in accelerating the pathology of osteoarthritis (OA). The pathways regulating joint calcification, in particular cartilage calcification, are not completely understood, but inflammation and the formation of reactive oxygen species (ROS) are contributory factors. The xanthine oxidase (XO) form of xanthine oxidoreductase (XOR), the key enzyme in xanthine and uric acid metabolism, is a major cellular source of superoxide. We hypothesized that XOR could be implicated in chondrocyte mineralization and cartilage calcification and degradation in OA. We showed both in murine primary chondrocyte and chondrogenic ATDC5 cells, that mineralization was inhibited by two different XOR inhibitors, febuxostat and allopurinol. In addition, XOR inhibition reduced the expression of the pro-mineralizing cytokine interleukin-6 (IL-6). We next generated XOR knock-out chondrocyte cell lines with undetectable XOR expression and XO activity. XOR knock-out chondrocyte cells showed decreased mineralization and reduced alkaline phosphatase (Alp) activity. To assess the precise form of XOR involved, primary chondrocytes of XOR mutant mice expressing either the XDH form (XDH ki) or the XO form (XO ki) were studied. We found that XO ki chondrocytes exhibited increased mineralization compared to XDH ki chondrocytes, and this was associated with enhanced Alp activity, ROS generation and IL-6 secretion. Finally, we found increased XOR expression in damaged vs. undamaged cartilage obtained from OA patients and XOR expression partially co-localized with areas showing pathologic calcification. Altogether, our results suggest that XOR, via its XO form, contribute to chondrocyte mineralization and pathological calcification in OA cartilage.

Self‐Resistance in the Biosynthesis of Fungal Macrolides Involving Cycles of Extracellular Oxidative Activation and Intracellular Reductive Inactivation

AbstractSelf‐resistance genes are employed by many microbial producers of bioactive natural products to avoid self‐harm. Herein, we describe a unique strategy for self‐resistance toward a macrolide antibiotic, A26771B (1), identified by elucidating its biosynthetic pathway in the fungus Penicillium egyptiacum. A highly reducing polyketide synthase and a trans‐acting thioesterase generate the macrolide backbone, and a cytochrome P450 and an acyltransferase, respectively catalyze hydroxylation and succinylation to form the prodrug berkeleylactone E (2). Then, extracellular oxidative activation by a secreted flavin‐dependent oxidase forms 1, while intracellular reductive inactivation by a short‐chain reductase reforms 2, forming a redox cycle. Our work illustrates a unique redox‐mediated resistance mechanism for fungal antibiotics and contributes to the understanding of antibiotic biosynthesis and resistance.

Self-Resistance in the Biosynthesis of Fungal Macrolides Involving Cycles of Extracellular Oxidative Activation and Intracellular Reductive Inactivation.

Self-resistance genes are employed by many microbial producers of bioactive natural products to avoid self-harm. Herein, we describe a unique strategy for self-resistance toward a macrolide antibiotic, A26771B (1), identified by elucidating its biosynthetic pathway in the fungus Penicillium egyptiacum. A highly reducing polyketide synthase and a trans-acting thioesterase generate the macrolide backbone, and a cytochrome P450 and an acyltransferase, respectively catalyze hydroxylation and succinylation to form the prodrug berkeleylactone E (2). Then, extracellular oxidative activation by a secreted flavin-dependent oxidase forms 1, while intracellular reductive inactivation by a short-chain reductase reforms 2, forming a redox cycle. Our work illustrates a unique redox-mediated resistance mechanism for fungal antibiotics and contributes to the understanding of antibiotic biosynthesis and resistance.

Genetics and epigenetics of pelvic organs prolapse

Identification of the genetic variants underlying the heredity of pelvic organ prolapse helps to identify useful markers for assessing clinical risk, prognosis and response to treatment. An association of polymorphisms in the ADRB 3 gene with an overactive bladder and in the COL1A1 gene with PTO was detected. AA mutation of the COL3A1 rs1800255 genotype increases the probability of VET by 4.8 times. There is evidence of involvement in the development of PTO genes of the extracellular matrix degradation group (ECM), which includes genes encoding matrix metalloproteinases. During the study of diseases of the connective tissue, their syntropy was revealed, which is genetically determined as a common risk haplotype of the FBLN 5 gene. The significance of epigenetic mechanisms for the development of VET is shown in the form of lysyl oxidase (LO) gene suppression. So far, the role of factors that induce changes in epigenetic regulation and lead to the appearance of clinical symptoms of prolapse is not clear.

A Phase II Non-Randomized Study to Evaluate the Efficacy of Single Dose Rasburicase (1.5mg) in Adult Acute Leukemia and High Grade Lymphomas with Established Tumor Lysis Syndrome

BACKGROUND Tumor lysis syndrome (TLS) refers to a constellation of metabolic derangements due to the breakdown of tumor cells.It is a medical emergency and is associated with significant morbidity and mortality. Hyperuricemia is a key manifestation of TLS, that can lead to renal insufficiency and trigger a vicious cycle of metabolic abnormalities. Rapid resolution of hyperuricemia is key to successful outcomes. Rasburicase is a recombinant form of enzyme urate oxidase, approved in patients with established TLS,which converts the uric acid to soluble allantoin. It acts rapidly, bringing down the levels in as less as 4 hours. The approved dose is 0.2 mg/kg/day for 5 days. While there is efficacy data to support the use of fixed low-dose (1.5-3 mg) rasburicase in the prophylactic setting,no such data exists in the therapeutic setting. Each 1.5 mg vial costs about 8,000 rupees (115 USD), the dose required for a 60 kg adult would be 12 mg, which equals to about 300,000 rupees (4340 USD) for 5 days. Given the cost constraints, most of our patients with established TLS receive only a single fixed dose of rasburicase at 1.5 mg as a standard of care. We have observed that even this lower dose is adequate to normalize the uric acid levels in a majority of them. We planned to prospectively evaluate the efficacy of single-dose rasburicase in patients with established TLS. METHODS We conducted a single centre phase II study and enrolled patients (>15 years) with de-novo acute leukemia and high-grade lymphoma with hyperuricemia(laboratory or clinical TLS) and ECOG PS ≤ 4. Patients with hemodynamic instability or neurological impairment were excluded. After enrollment, patients received rasburicase at a dose of 1.5 mg fixed-dose intravenous over 30 min. Patients were monitored daily either as an in- or outpatient. Blood samples to measure the renal function tests and electrolytes were collected at 4 hours, 24-h, 48-h, Day 3, Day 4, and Day 5. Blood samples were transported in heparinized tubes at room temperature and processed immediately. Additional 1.5 mg dose of rasburicase was given if the plasma uric acid level did not decline by at least 50% at 24 hours. Additional doses were also given at the clinician's discretion in case of rising uric acid levels on subsequent monitoring. The primary objective was to estimate the rate of plasma uric acid response (Defined as normalization of uric acid at 48 hours that is sustained till day 5). Secondary objectives were to assess the number of patients requiring additional doses of rasburicase, need for dialysis, mortality attributable to TLS and adverse events due to rasburicase. The sample size was calculated using Simon's two-stage design.52 patients were required to detect a Plasma UA response rate of 85% (Compared to 99% with the approved dose) with a type I error of 5% and power of 80%. Due to a higher than anticipated attrition rate, we enrolled 70 patients. Results: A total of 70 patients were enrolled in the study, 60 were analyzed for secondary and 53 for the primary endpoint (Fig 1). The baseline features are summarized.(Fig 1) Plasma uric acid normalized at 48 hours in 37/53 (70%) of the patients and became normal at day 5 in 46/53(87%) of the patients. The corresponding figures for the entire cohort were 68% and 82% respectively. Only 3 patients required hemodialysis. 11 patients were managed as outpatient, the remaining were admitted. 3 patients required ICU admission. 20 patients required additional doses of rasburicase, out of which 13 patients required 2 doses, 5 patients required 3 doses, 1 each required 4 and 5 doses. The total number of vials required in the study were 83 vials. In comparison, 2120 vials would have been required for a cohort of people weighing 60kgs. Hypersensitivity reaction, cardiac dysfunction, methemoglobinemia were not seen in any patient. 1 patient developed hemolysis post rasburicase. Other adverse events were fever(25%), vomiting(10%), abdominal pain(13%). All 60 evaluable patients were alive at the end the study. Conclusion: Lower dose of rasburicase is sufficient and cost-effective in established TLS. This strategy obviates the need for FDA approved higher doses. Disclosures No relevant conflicts of interest to declare.

Exploration of vascular adhesion protein-1 expression in patients with conjunctivitis associated systemic lupus erythematosus using 2D-DIGE.

Conjunctivitis associated systemic lupus erythematosus (caSLE) is a connective tissue autoimmune disease with a large spectrum of clinical manifestations. The disease may lead to ocular complications and in severe cases may be sight-threatening. This study investigated the expression of vascular adhesion protein-1 (VAP-1) in SLE patients with conjunctivitis and the relationship between VAP-1 and other proteins in the onset of the disease. Ten patients with caSLE (caSLE group) and 10 healthy volunteers (control group) were enrolled in the study, in order to evaluate the VAP-1 expression levels in blood. Protein expression profiling was performed with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and analyzed with matrix-assisted laser desorption/deionization time-of-flight (MALDI-TOF) used in tandem with mass spectrometry. In the caSLE group, 8 proteins were expressed differenty compared with the control group: C-reactive protein, hemoglobin subunit β, VAP-1, A-albumin (AFAM), enolase and immunoglobulin heavy constant mu were upregulated; interferon regulatory factor-1 and serum amyloid A2 protein were downregulated. Western blotting results are consistent with the proteomics results, showing that in caSLE group VAP-1 expression is increased in comparison to the control group. VAP-1 protein participates in the inflammatory reaction in the form of amine oxidase, copper containing 3. Bioinformatics analysis suggested that VAP-1 protein could be used as a candidate protein for further study regarding its role as a potential protein marker for screening and effectively monitoring caSLE.

π-π Stacking Interaction in an Oxidized CuII -Salen Complex with a Side-Chain Indole Ring: An Approach to the Function of the Tryptophan in the Active Site of Galactose Oxidase.

In order to gain new insights into the effect of the π-π stacking interaction of the indole ring with the CuII -phenoxyl radical as seen in the active form of galactose oxidase, we have prepared a CuII complex of a methoxy-substituted salen-type ligand, containing a pendent indole ring on the dinitrogen chelate backbone, and characterized its one-electron-oxidized forms. The X-ray crystal structures of the oxidized CuII complex exhibited the π-π stacking interaction of the indole ring mainly with one of the two phenolate moieties. The phenolate moiety in close contact with the indole moiety showed the characteristic phenoxyl radical structural features, indicating that the indole ring favors the π-π stacking interaction with the phenoxyl radical. The UV/Vis/NIR spectra of the oxidized CuII complex with the pendent indole ring was significantly different from those of the complex without the side-chain indole ring, and the absorption and CD spectra exhibited a solvent dependence, which is in line with the phenoxyl radical-indole stacking interaction in solution. The other physicochemical results and theoretical calculations strongly support that the indole ring, as an electron donor, stabilizes the phenoxyl radical by the π-π stacking interaction.

A new crystal form of Aspergillus oryzae catechol oxidase and evaluation of copper site structures in coupled binuclear copper enzymes

Coupled binuclear copper (CBC) enzymes have a conserved type 3 copper site that binds molecular oxygen to oxidize various mono- and diphenolic compounds. In this study, we found a new crystal form of catechol oxidase from Aspergillus oryzae (AoCO4) and solved two new structures from two different crystals at 1.8-Å and at 2.5-Å resolutions. These structures showed different copper site forms (met/deoxy and deoxy) and also differed from the copper site observed in the previously solved structure of AoCO4. We also analysed the electron density maps of all of the 56 CBC enzyme structures available in the protein data bank (PDB) and found that many of the published structures have vague copper sites. Some of the copper sites were then re-refined to find a better fit to the observed electron density. General problems in the refinement of metalloproteins and metal centres are discussed.