Iodonitrotetrazolium Research Articles

Antibiofilm Activity of Extract and a Compound Isolated from Triumfetta welwitschii against Pseudomonas aeruginosa.

Triumfetta welwitschii has been used as a traditional medicine in Africa. It is documented as a rich source of phytochemicals with antibacterial activities. To further explore the antibacterial potential of these phytochemical components, the phytochemical profile of the dichloromethane: methanol leaf extract from T. welwitschii was investigated using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Compounds were isolated from the extract using column chromatography and thin-layer chromatography. Compound B1 was isolated from the fraction eluted by 90 hexane:10 ethyl acetate using column chromatography. The antibacterial activity of B1 against Pseudomonas aeruginosa was evaluated in vitro using the broth microdilution method and the iodonitrotetrazolium (INT) colorimetric assay. The antibiofilm activities of the extract and B1 against P. aeruginosa were determined by quantifying the biofilms using crystal violet. The effect of the extract and B1 on capsular polysaccharide and extracellular DNA content of biofilm formed by P. aeruginosa was determined using phenol-sulphuric acid and propidium iodide, respectively. A total of 28 peaks were detected and identified using UPLC-MS/MS. The three most abundant phytochemicals identified were catechin, umbelliferone, and a luteolin derivative. B1 showed antibacterial activity against P. aeruginosa with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) value of 25 μg/ml. Only 38% and 6% of the biofilms were formed in the presence of the extract and B1, respectively. The extract and B1 reduced the capsular polysaccharide content in biofilms formed in P. aeruginosa by 40% and 65%, respectively. The extract and B1 significantly reduced the extracellular DNA content of biofilms by 29% and 72%, respectively. The results of this study provide evidence of the antibacterial and antibiofilm activities of B1 and leaf extracts from T. welwitschii. Future work should identify the chemical structure of B1 using nuclear magnetic resonance and mass spectrometry.

Validation of the in vivo Iodo-Nitro-Tetrazolium (INT) Salt Reduction Method as a Proxy for Plankton Respiration

Knowledge of the magnitude and variability of plankton respiration is a crucial gap in our understanding of marine carbon cycling. In order to validate the INT reduction method as a proxy for plankton respiration, we have compiled and analysed a dataset (n = 376) of concurrent measurements of dissolved oxygen consumption (CRO2) and in vivo reduction of 2-para (iodophenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride tetrazolium salt (INT) spanning a wide range of oceanic regions and physicochemical conditions. Data were randomly divided into two independent subgroups: two thirds of the data were used to derive a regression conversion between dissolved oxygen consumption and INT reduction (“training” dataset) and one third of the data was used to validate the regression (“test” dataset). There was a significant relationship between the log-transformed dissolved oxygen consumption rates and the log-transformed INT reduction rates (INTT) with the “training” dataset (logCRO2 = 0.72logINTT + 0.44, R2 = 0.69, n = 249, p 8 ˚C and chlorophyll-a concentrations >0.2 µg L-1 and reduced at lower temperatures and chlorophyll-a concentrations. The results of this study endorse the adequate performance and reliability of the INT method for natural plankton communities.

Fractionation of Heteromorpha arborescens var abyssinica (Apiaceae) leaf extracts based on polarity leads to a marked change in cytotoxicity that may yield a commercially useful product

Parasitic gastroenteritis caused by helminths remains a major cause of mortality and morbidity especially in small ruminants in the tropics. Haemonchus contortus is the major helminth responsible for this disease. Chemical anthelmintics are the most reliable means of treatment. The frequent use of these anthelmintics has led to resistance development. The issue of residues in meat and milk also motivates the use of plants and plant derived products as alternate to chemical anthelmintics. The anthelmintic, antimicrobial activity and cytotoxicity of the fractions of Heteromorpha arborescens var abyssinica were investigated. The egg hatch assay and the larval development test were used to determine the anthelmintic activity. A serial microdilution method with iodo nitrotetrazolium violet as growth indicator was used to determine the antibacterial and antifungal activities. The cellular cytotoxicity of the fractions was determined using the MTT assay. The egg hatch assay led to an EC50 of 0.68, 0.73, 1.58 and 2.88mg/ml for the butanol, ethyl acetate, chloroform and hexane fractions respectively. The larval development test of the butanol, chloroform, ethyl acetate and hexane fractions led to EC50 values of 0.64, 1.05, 1.05 and 3.28mg/ml respectively. Because extracts or fractions can be more useful if they have activity against more than one indication the antimicrobial activity of the fractions was also determined. The best antifungal minimum inhibitory concentrations (MICs) of 0.08mg/ml was against Cryptococcus neoformans and 0.63mg/ml against Aspergillus fumigatus and Candida albicans. Pseudomonas aeruginosa was most susceptible bacterium in this study. The cytotoxicity LC50 values of the fractions of hexane, ethyl acetate, butanol and chloroform were 1.40, 0.91, 2.06 and 2.04mg/ml. Fractionation of the acetone extract of H. arborescens var abyssinica led to a remarkable 35 to 50-fold reduction in cytotoxicity of the fractions compared to the original extract.The anthelmintic activities of the crude leaf extract and fractions were not that high, but due to the decreased cytotoxicity the butanol fraction had a three-fold higher efficacy against H. contortus in both assays than toxicity against the cells. This may lead to a product that can be used to treat helminthiasis in animals if animal safety is in line with cellular safety. The results stress the importance of determining the cytotoxicity of plant extracts with a potential to be used in animal studies. The selectivity index may be more important than the activity of an extract against microorganisms or parasites.

Antibacterial and Antioxidant Activity of Extracts from Selected Probiotic Bacteria

<p>Probiotic extracts can potentially be used as bio-preservatives and in reduction of oxidative stress. The study investigated the antibacterial and antioxidant activity of methanol extracts from freeze-dried cells of probiotic <em>Lactobacillus</em> strains identified using molecular techniques. The quantitative microplate method, which employed <em>p</em>-iodonitrotetrazolium (INT) and the method by Brand-Williams et al. (1995) were employed to investigate quantitatively the antibacterial and the antioxidant activity, respectively, of probiotic extracts. The MIC values extracts from most probiotic strains, tested against indicator bacterial pathogens, were in the range of 1.25 – 5 mg/mL while that of <em>Lb. casei</em> strain B and <em>Lc. lactis</em> subsp <em>lactis</em> strain X was at least 20 mg/mL after 24 h of incubation at 37°C. At the highest extract concentration of 20 mg/mL used in the study, <em>Lb. acidophilus</em>, <em>Lb. rhamnosus</em> and <em>Lb. casei</em> strains had 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities of 77.9 - 86.1%, 45.7 - 86.4% and 36.9 – 45.8% respectively. Quantitative antibacterial and antioxidant activities of methanol extracts from freeze-dried cells of probiotic <em>Lactobacillus</em> strains was determined for the first time.</p>

Hexavalent Chromium-Induced Apoptosis in Rat Uterus: Involvement of Oxidative Stress

The present study is designed to test the hypothesis that oxidative stress mediates hexavalent chromium (VI)–induced apoptosis in uterus. Female Wistar rats received an intraperitoneal (i.p.) injection of potassium dichromate at doses of 1 and 2 mg/kg. Superoxide anion production was assessed by determination of the reduction of cytochrome c and iodonitrotetrazolium (INT), lipid peroxidation (LPO), metallothioneins (MTs), and catalase (CAT) activity. The expression of Bax and Bcl-2 proteins was investigated. After 15 days of treatment, an increase of LPO and MT levels occurred, whereas CAT activity decreased. Intense apoptosis was observed in endometriotic stromal cells of Cr-exposed rats. Bax protein expression was induced in endometriotic stromal cells with 1 mg of Cr(VI)/kg, and in stromal and epithelial cells at the higher dose. These results clearly suggest that Cr(VI) subacute treatment causes oxidative stress in rat uterus, leading to endometriotic stromal cells apoptosis.

A blue native polyacrylamide gel electrophoretic technology to probe the functional proteomics mediating nitrogen homeostasis in Pseudomonas fluorescens

As glutamate and ammonia play a pivotal role in nitrogen homeostasis, their production is mediated by various enzymes that are widespread in living organisms. Here, we report on an effective electrophoretic method to monitor these enzymes. The in gel activity visualization is based on the interaction of the products, glutamate and ammonia, with glutamate dehydrogenase (GDH, EC: 1.4.1.2) in the presence of either phenazine methosulfate (PMS) or 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium (INT). The intensity of the activity bands was dependent on the amount of proteins loaded, the incubation time and the concentration of the respective substrates. The following enzymes were readily identified: glutaminase (EC: 3.5.1.2), alanine transaminase (EC: 2.6.1.2), aspartate transaminase (EC: 2.6.1.1), glycine transaminase (EC: 2.6.1.4), ornithine oxoacid aminotransferase (EC: 2.6.1.13), and carbamoyl phosphate synthase I (EC: 6.3.4.16). The specificity of the activity band was confirmed by high pressure liquid chromatography (HPLC) following incubation of the excised band with the corresponding substrates. These bands are amenable to further molecular characterization by a variety of analytical methods. This electrophoretic technology provides a powerful tool to screen these enzymes that contribute to nitrogen homeostasis in Pseudomonas fluorescens and possibly in other microbial systems.

Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4±1.7nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5±2.8nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26±2.6nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48±20.2nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the importance of this domain.

Role of Putative Second Transmembrane Region of Nox2 Protein in the Structural Stability and Electron Transfer of the Phagocytic NADPH Oxidase

Flavocytochrome b(558) (cytb) of phagocytes is a heterodimeric integral membrane protein composed of two subunits, p22(phox) and gp91(phox). The latter subunit, also known as Nox2, has a cytosolic C-terminal "dehydrogenase domain" containing FAD/NADPH-binding sites. The N-terminal half of Nox2 contains six predicted transmembrane α-helices coordinating two hemes. We studied the role of the second transmembrane α-helix, which contains a "hot spot" for mutations found in rare X(+) and X(-) chronic granulomatous disease. By site-directed mutagenesis and transfection in X-CGD PLB-985 cells, we examined the functional and structural impact of seven missense mutations affecting five residues. P56L and C59F mutations drastically influence the level of Nox2 expression indicating that these residues are important for the structural stability of Nox2. A53D, R54G, R54M, and R54S mutations do not affect spectral properties of oxidized/reduced cytb, oxidase complex assembly, FAD binding, nor iodonitrotetrazolium (INT) reductase (diaphorase) activity but inhibit superoxide production. This suggests that Ala-53 and Arg-54 are essential in control of electron transfer from FAD. Surprisingly, the A57E mutation partially inhibits FAD binding, diaphorase activity, and oxidase assembly and affects the affinity of immunopurified A57E cytochrome b(558) for p67(phox). By competition experiments, we demonstrated that the second transmembrane helix impacts on the function of the first intracytosolic B-loop in the control of diaphorase activity of Nox2. Finally, by comparing INT reductase activity of immunopurified mutated and wild type cytb under aerobiosis versus anaerobiosis, we showed that INT reduction reflects the electron transfer from NADPH to FAD only in the absence of superoxide production.

New Enzymatic Colorimetric Method for the Quantitative Determination of Phenylalanine in Dry-Blood Spots

Phenylketonuria (PKU) is an autosomal recessive disorder, which is characterized by severe mental retardation, microcephaly and seizures. The symptoms of this disease can be prevented if detected soon after birth. Therefore, blood Phenylalanine (Phe) measurement is essential for the early diagnosis, treatment and dietary monitoring of PKU patients. The goal of this research was to introduce a rapid, precise and effective enzymatic colorimetric method using the recombinant Bacillus badius phenylalanine dehydrogenase (PheDH; EC 1.4.1.20) for the quantitative determination of Phe in dry-blood spots. This test was based on the enzymatic reaction of PheDH coupled with an artificial electron acceptor system composed of phenazine methosulfate (PMS) and iodonitro tetrazolium chloride (INT). This assay system contained PMS to transfer the electrons of NADH to INT, enabling the formation of formazan with an absorbance at 490 nm. Calibration curve was plotted and the experimental data were fitted by linear regression analyze. The regression equation and correlation coefficient (R 2 ) were Y= 0.0109x + 0.032 and R 2 =0.996, respectively. This method showed a recovery in the range of 95.1%-102.6% and had the limit detection of 0.5 mg/dl for Phe. The between run coefficients of variation (CVs) mean was between 3.8% and 9.1%. The within-run CVs was between 8.5% and 18.6%. Furthermore, no interferences from other amino acids and Phe derivatives were observed. Altogether, we here presented a quick and reliable enzymatic colorimetric assay for application in newborn screening and monitoring of PKU patients.

Two Atypical l-Cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, l-Cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba histolytica

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular L-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and L-cystine to L-cysteine and also function as ferric and ferredoxin-NADP(+) reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower K(m) and higher k(cat)/K(m) values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred L-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, L-cysteine/L-cystine homeostasis, iron reduction, and the activation of metronidazole.

Inhibition of NADPH oxidase by apocynin inhibits lipopolysaccharide (LPS) induced up-regulation of arginase in rat alveolar macrophages

Reactive oxygen species participate in the pathogenesis of inflammatory airway diseases, in which increased arginase may play a role by interfering with nitric oxide (NO) synthesis and providing substrate for collagen synthesis. Therefore a modulatory role of reactive oxygen species for arginase was explored in alveolar macrophages using the NADPH oxidase inhibitor apocynin. The effects of lipopolysacharides (LPS) and apocynin on nitrite accumulation, arginase activity and mRNA for inducible NO synthase (iNOS), arginase I and II were determined. Superoxide anion (O 2 −) release was analysed by the iodonitrotetrazolium (INT) formazan assay. LPS (1 μg/ml) caused a 55%, transient increase in INT formation, i.e. O 2 − release which was inhibited by apocynin (500 μM). LPS caused a 2 fold increase in arginase activity and a marked increase in mRNA encoding arginase I, the predominant isoenzyme. Both effects were largely attenuated by apocynin. Apocynin did not affect the stability of arginase I mRNA, but accelerated the decline of arginase activity when protein synthesis was inhibited by cycloheximide. Apocynin also reduced LPS-induced nitrite accumulation (by 30%) and iNOS mRNA expression, but the magnitude of these effects was smaller than that on arginase I. Arginase I mRNA was also increased following exposure to hydrogen peroxide (H 2O 2, 200 μM). In conclusion, inhibition of NADPH oxidase in alveolar macrophages causes down-regulation of arginase, indicating that reactive oxygen species exert stimulatory effects on arginase. Enhanced transcription of arginase mRNA and prolongation of the life time of the active enzyme appear to contribute to the enhanced arginase activity.

In situ localization of glucose and sucrose in dehydrating leaves of Sporobolus stapfianus

Accumulation of soluble carbohydrates during dehydration stress is thought to be a very important mechanism for the acquisition of desiccation tolerance. Despite the proposed importance of soluble carbohydrate accumulation (especially sucrose), nothing is known about the cellular localization of carbohydrates in desiccation-tolerant plants. The present study proposes a novel and selective method for the in situ localization of sucrose and glucose in the desiccation-tolerant plant Sporobolus stapfianus. The detection of sucrose and glucose is based on a series of coupled enzymatic reactions leading to the formation of NADH. Iodonitrotetrazolium (INT) reacts with NADH, thereby providing the red-colored insoluble INT-formazan. Stained tissue sections were immediately visualized using light microscopy. Localization of the respective sugars was site specific. Sucrose was visualized in all leaf cell types during dehydration: vascular bundles, bundle sheath cells, mesophyll cells and epidermal cells. Similarly, glucose was shown to be localized in the same leaf compartments as reported for sucrose. This is the first report that describes sucrose localization in dehydrating leaf tissues of a "resurrection" plant. We conclude that, during dehydration stress, sucrose accumulates in all viable tissues; these results are in agreement with the previously proposed theories about its function as a cellular protectant.

Leu505 of Nox2 is crucial for optimal p67phox-dependent activation of the flavocytochromeb558 during phagocytic NADPH oxidase assembly

The role of Leu505 of Nox2 on the NADPH oxidase activation process was investigated. An X-CGD PLB-985 cell line expressing the Leu505Arg Nox2 mutant was obtained, exactly mimicking the phenotype of a previously published X91+-CGD case. In a reconstituted cell-free system (CFS), NADPH oxidase and iodonitrotetrazolium (INT) reductase activities were partially maintained concomitantly with a partial cytosolic factors translocation to the plasma membrane. This suggests that assembly and electron transfer from NADPH occurred partially in the Leu505Arg Nox2 mutant. Moreover, in a simplified CFS using purified mutant cytochrome b558 and recombinant p67phox, p47phox, and Rac1proteins, we found that the Km for NADPH and for NADH was about three times higher than those of purified WT cytochrome b558, indicating that the Leu505Arg mutation induces a slight decrease of the affinity for NADPH and NADH. In addition, oxidase activity can be extended by increasing the amount of p67phox in the simplified CFS assay. However, the maximal reconstituted oxidase activity using WT purified cytochrome b558 could not be reached using mutant cytochrome b558. In a three-dimensional model of the C-terminal tail of Nox2, Leu505 appears to have a strategic position just at the entry of the NADPH binding site and at the end of the alpha-helical loop (residues 484-504), a potential cytosolic factor binding region. The Leu505Arg mutation seems to affect the oxidase complex activation process through alteration of cytosolic factors binding and more particularly the p67phox interaction with cytochrome b558, thus affecting NADPH access to its binding site.

Crucial Role of Two Potential Cytosolic Regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH Oxidase Activation

Assembly of cytosolic factors p67(phox) and p47(phox) with cytochrome b(558) is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop (191)TSSTKTIRRS(200) and the C-terminal (484)DESQANHFAVHHDEEKD(500) of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47(phox) translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop(Nox4)-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the alpha-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47(phox) membrane translocation. This suggests that the alpha-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.

Functional Analysis of NADPH Oxidase in Granulocytic Cells Expressing a ▵488-497 gp91phox Deletion Mutant

Chronic granulomatous disease (CGD) is a group of inherited disorders in which phagocytes are unable to generate superoxide (O(2)(-)) due to genetic defects in any 1 of 4 essential NADPH oxidase components. Mutations in the X-linked gene for gp91(phox), the large subunit of the flavocytochrome b(558) heterodimer, account for the majority of CGD. An X-CGD patient in which a splice junction mutation results in an in-frame deletion of 30 nucleotides encoding amino acids 488 to 497 of gp91(phox) (delta488-497 gp91(phox)) has previously been reported. In this study, we generated myeloid PLB-985 cells expressing the mutant triangle delta488-497 gp91(phox) to further characterize its functional properties. These cells mimicked the phenotype of the patient's neutrophils with normal expression of a nonfunctional delta488-497 gp91(phox) flavocytochrome. Translocation of p47(phox) and p67(phox) to delta488-497 gp91(phox) PLB-985 plasma membranes was not affected, as determined both in activated intact cells and in the cell-free system. Furthermore, a synthetic peptide corresponding to residues 488-497 of gp91(phox) was relatively ineffective in inhibiting O(2)(-) production in the cell-free oxidase assay (IC50, approximately 500 micromol/L), suggesting that residues 488-497 of gp91(phox) are not directly involved in oxidase assembly. Mutant delta488-497 gp91(phox) flavocytochrome failed to support iodonitrotetrazolium (INT) reduction, showing a disruption of electron transfer from NADPH to the FAD center of gp91(phox). However, the FAD binding capacity of the mutant flavocytochrome was normal, as measured by equilibrium dialysis. Taken together, these results suggest that the delta488-497 deletion in gp91(phox) disrupts electron transfer to FAD, either due to a defect in NADPH binding or to impaired delivery of electrons from NADPH.

Quantificação de micélio extrarradicular de fungos micorrízicos arbusculares em plantas cítricas e endófitos: I. Método empregado

Otimização de métodos de extração e avaliação de micélio extrarradicular ativo (MEA) e total (MET) constituem os principais passos para um estudo mais profundo da interação fungo micorrízico arbuscular (FMA) e hospedeiro, já que a absorção e translocação dos nutrientes do substrato ao hospedeiro são realizadas, respectivamente, por tais estruturas fúngicas. Métodos de extração de micélio extrarradicular de FMAs propostos por diversos autores foram reavaliados e mesclados, originando um método exclusivo e de fácil execução, juntamente com a calibração de métodos para avaliação e quantificação de MEA (fluorescência induzida com a hidrólise do diacetato de fluoresceína e redução de iodonitrotetrazólio), em substrato arenoso.

Enzymatic method for assaying uric acid in serum with a new tetrazolium salt produces water-soluble formazan dye.

To apply an enzymatic method for assaying uric acid in serum based on the uricase (EC 1.7.3.3)-catalase (EC 1.11.1.6)-formaldehyde dehydrogenase (FADH, EC 1.2.1.46) coupled with the new tetrazolium salt producing a water-soluble formazan dye as an indicator system. Unlike the traditional tetrazolium salts, e.g., iodonitrotetrazolium (INT) and nitrotetrazolium blue (NTB), the corresponding formazan dye produced did not absorb to the test tube and the reaction cells of the analyzer. Moreover, this formazan dye had a higher water solubility and more sensitivity. A two electron reduction of tetrazolium salt with NADH, produced by the uricase-catalase-FADH reaction, was mediated by an electron carrier, 1-methoxy PMS. The generation of formazan, monitored at 440 nm, was proportional to the concentration of uric acid in serum. The sensitivity of this method was about 1.5 times that of the peroxidase-TOOS [N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine] method. The assay was evaluated with TBA-80FR.NEO biochemical analyzer. The average within-run and between-day imprecision (CV) were 0.75-2.44% and 3.20-3.33%, respectively. Results of the proposed method (y) correlated well with those determined by the conventional chromogen method (x): y = 0.970 x - 0.018 mmol/L (Sy l x = 0.019 mmol/L, r = 0.993, n = 67). We also present data showing that the method is rapid, relatively free of interference, and amenable to automation.

Production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes from endrin-treated rats

Recent studies have shown that the adminstration of endrn to rodents induced lipid peroxidation in various tissues and decreases glutathione content. These results suggest that endrin produces reactive oxygen species and/or free radicals. We have therefore examined the effects of endrin(4.5 mg/kg) on the production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes in rats. The effects of endrin on hepatic mitochondrial and microsomal lipid peroxidation and membrane fluidity as well as the incidence of hepatic nuclear DNA damage were also examined. Twenty-four hours after endrin administration, significant increases in the production of the chemiluminescence by the three tissue fractions were observed. Furthermore, peritoneal macrophages form endrin-animals resulted in 3.0- and 2.8-fold increases in cytochrome c and iodonitrotetrazolium (INT) reduction, indicating enhanced production of superoxide anion. Endrin administration also resulted in significant increases in lipid peroxidation of mitochondrial and microsomal membranes as well as decreases in the fluidity of these two membranous fractions. A significant increase in hepatic nuclear DNA single-strand breaks also occurred in response to endrin administration. The reslts indicate that macrophage, mitochondria, and microsomes produce reactive oxygen species following endrin administration, and these reactive oxygen species may contribute to the toxic manifestations of endrin.

Characterization of an inducible, membrane-bound iminodiacetate dehydrogenase from Chelatobacter heintzii ATCC 29600

Iminodiacetate (IDA) is a xenobiotic intermediate common to both aerobic and anaerobic metabolism of nitrilotriacetate (NTA). It is formed by either NTA monooxygenase or NTA dehydrogenase. In this paper the detection and characterization of a membrane-bound iminodiacete dehydrogenase (IDA-DH) from Chelatobacter heintzii ATCC 29600 is reported, which oxidizes IDA to glycine and glyoxylate. Out of 15 compounds tested, IDA was the only substrate for the enzyme. Optimum activity of IDA-DH was found at pH 8.5 and 25°C, respectively, and the Km for IDA was found to be 8mM. Activity of the membrane-bound enzyme was inhibited by KCN, antimycine and dibromomethylisopropyl-benzoquinone. When inhibited by KCN IDA-DH was able to reduce the artificial electron acceptor iodonitrotetrazolium (INT). It was possible to extract IDA-DH from the membranes with 2% cholate, to reconstitute the enzyme into soybean phospholipid vesicles and to obtain IDA-DH activity (more than 50% recovery) using ubiquinone Q1 as the intermediate electron carrier and INT as the final electron acceptor. Growth experiments with different substrates revealed that in all NTA-degrading strains tested both NTA monooxygenase and IDA-DH were only expressed when the cells were grown on NTA or IDA. Furthermore, in Cb. heintzii ATCC 29600 growing exponentially on succinate and ammonia, addition of 0.4 g l-1 NTA led to the induction of the two enzymes within an hour and NTA was utilized simultaneously with succinate. The presence of IDA-DH was confirmed in ten different NTA-degrading strains belonging to three different genera.

A convenient and inexpensive chemo-radiosensitivity assay for lung cancer cells using Terasaki's microplate.

We devised a simple in vitro sensitivity test for lung cancer cells using Terasaki's microplate. We used the test to screen for sensitivity to various carcinostatic drugs and radiation, and to determine the optimum method of administration. This assay has been used in routine clinical examinations because about 40% of non-small cell carcinoma and 80% of small cell carcinoma of the lung can be subcultured. We describe here our patients who underwent treatment, various sensitivity tests and the preparation of an optimal course of treatment based upon the results of the sensitivity tests. Cells were placed in primary culture as previously described for short-term selective culture, and 2nd-3rd generation subcultured cells were transferred to individual wells of Terasaki's microplates for various sensitivity tests. After culture for 10 days, the effect was evaluated using 0.1% iodonitrotetrazolium (INT). This test permits a variety of sensitivity tests and various studies of clinical models of intensive treatment to be performed conveniently and reproducibly, because subcultured cancer cells are used. Another advantage is that these cells can be applied to basic investigations, including the preparation of monoclonal antibodies and chromosomes, DNA ploidy and oncogene studies.