Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms

Lysine-ketoglutaratc reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses L-lysine and α-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and thereafter decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to α-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibriumordered binding mechanism with a binding order of lysine, α-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.

Isolation of ergosterol peroxide and its reversion to ergosterol in the pathogenic fungus Sporothrix schenckii

Ergosterol peroxide, a presumed product of the H2O2-dependent enzymatic oxidation of ergosterol, has been isolated from yeast from yeast forms of the pathogenic fungus Sporothrix schenckii. The substance, which may have a role in fungal virulence, has been characterized mainly using spectroscopic methods (1H and 13C nuclear magnetic resonance and high resolution mass spectra). The purified compound showed a molecular formula of C28H44O3, displaying characteristic features of epidioxy sterols and was reverted to ergosterol when submitted to S. schenckii enzymatic extract.

Sensor for hydrogen peroxide based on Prussian Blue modified electrode: Improvement of the operational stability

Improvement of the operational stability of amperometric sensors based on Prussian Blue (PB) modified glassy carbon electrodes is presented. The long term performance of the sensors was evaluated by injection of hydrogen peroxide (5 μM in potassium buffer) solutions in a flow-injection system during a period of 5-10 h. The following parameters were investigated and correlated with the performance of the sensor: the times for electrodeposition and electrochemical activation, temperature, storage time, pH, composition of the buffer solution and of volume sample injected. These analytical characteristics of the modified electrode can be emphasized: initial sensitivity of 0.3 A cm-2 M-1, detection limit of ca. 0.5 μM, precise results (r.s.d.< 1.5%) and possibility to carry out around 50 samples (50 μL) per hour.

Effects of bleaching with carbamide peroxide gels on microhardness of restoration materials

The objective of this in vitro study was to quantitatively assess the effects of bleaching with 10 and 15% carbamide peroxide (CP) on restoration materials by performing superficial microhardness analysis. Acrylic cylindrical containers (4 x 2 mm) were filled with the following restoration products: Charisma (Heraues Kulzer, Vila Santa Catarina, São Paulo, Brazil), Durafill VS (Heraeus Kulzer), Vitremer (3M, Sumaré, São Paulo, Brazil), Dyract (Dentsply, Petrópolis, Rio de Janeiro, Brazil), and Permite C (SDI, São Pauio, São Paulo, Brazil). Sixty samples were prepared of each restoration material. Twenty samples received bleaching treatment with 10% CP, 20 samples received bleaching treatment with 15% CP, and 20 samples were kept submerged in artificial saliva, which was replaced daily. The treatment consisted of immersion of the specimens in 1 cm3 of CP at 10 and 15% for 6 hours per day during 3 weeks, whereupon the test specimens were washed, dried, and kept immersed in artificial saliva for 18 hours. Then the test and control specimens were analyzed using a microhardness gauge. The Knoop Hardness Number (KHN) was taken for each test and control specimen at five different locations by applying a 25 g force for 20 seconds. The values obtained were transformed into KHNs and the mean was calculated. The data were submitted to statistical analysis by analysis of variance and Tukey test, p < .05. The means/standard deviations were as follows: Charisma: CP 10% 38.52/4.08, CP 15% 34.31/6.13, saliva 37.36/4.48; Durafill VS: CP 10% 18.65/1.65, CP 15% 19.38/2.23, saliva 18.27/1.43; Dyract AP: CP 10% 30.26/2.81, CP 15% 28.64/5.44, saliva 33.88/3.46; Vitremer: CP 10% 28.15/3.04, CP 15% 17.40/3.11, saliva 40.93/4.18; and Permite C: CP 10% 183.50/27.09, CP 15% 159.45/5.78, saliva 215.80/26.15. A decrease in microhardness was observed for the materials Dyract AP, Vitremer, and Permite C after treatment with CP at 10 and 15%, whereas no effect on either of the two composites (Charisma and Durafill) was verified. CLINICAL SIGNIFICANCE: The application of the carbamide peroxide gels at 10 and 15% did not alter the microhardness of the composite resins Charisma and Durafill. In situ and clinical studies are necessary to enable one to conclude that the reduction in microhardness of the materials effectively results in clinical harm to the restorations.

Use of 37% carbamide peroxide in the walking bleach technique: A case report

Dental bleaching represents an effective, conservative, and relatively low-cost method for improving the appearance of discolored pulpless teeth. Among the bleaching techniques, the walking bleach technique with sodium perborate associated with water or hydrogen peroxide stands out because of its esthetic results and safety. A modified walking bleach technique with the use of 37% carbamide peroxide as the bleaching agent is presented. Additionally, the adverse effects of dental bleaching in the following restorative procedures are discussed, showing the advantages with the use of 37% carbamide peroxide.

Diagnosis of 5α-reductase type 2 deficiency: Contribution of anti-Müllerian hormone evaluation

Aim: To evaluate anti-Müllerian hormone (AMH) levels in patients with clinical and molecular diagnosis of 5α-reductase 2 deficiency. Patients and methods: Data from 14 patients whose age ranged from 21 days to 29 years were analyzed according to age and pubertal stage. Sexual ambiguity was rated as Prader III in 11 patients. LH, FSH, testosterone (T), dihydrotestosterone (DHT) and AMH serum levels were measured in all but two patients, who had been previously submitted to gonadectomy; T and DHT were also measured in 20 age-matched controls. Results: Gonadotropin levels were normal in all but one patient who retained gonads (six of whom had reached puberty) and T/DHT ratio was elevated in all patients when compared to controls. All prepubertal patients had AMH levels < -1 SD for age, while most pubertal patients had AMH levels compatible with pubertal stage. Conclusions: Prepubertal patients with 5α-reductase 2 deficiency have AMH values in the lower part of the normal range. These data indicate that T does not need to be converted to DHT to inhibit AMH secretion by Sertoli cells. © Freund Publishing House Ltd., London.

Effect of 10% carbamide peroxide dental bleaching on microhardness of filled and unfilled sealant materials

Purpose: The purpose of this study was to quantitatively evaluate the effect of 10% carbamide peroxide on the microhardness of pit and fissure sealant materials. Methods: Fluroshield, Vitroseal Alfa, and one unfilled (Clinpro) sealants were placed in Teflon matrices (4 mm in diameter by 2 mm in height) and polymerized for 40 seconds. A total of 20 specimens were prepared for each material, in which half were assigned as the control group (stored in artificial saliva and no bleaching treatment). For the remaining half, Clarigel Gold bleaching agent (10% carbamide peroxide) was placed over the specimen surface for 4 hours/day during 4 weeks. When specimens were not under bleaching treatment, they were kept in artificial saliva. Afterwards, specimens were subjected to Knoop microhardness testing using a 25-g load for 5 seconds. Five measurements were made on the sealants' surfaces and then calculated in Knoop hardness values. The data were statistically analyzed by two-way analysis of variance and Tukey's tests with a 5% confidence level. Results: The results of this in vitro study showed that the application of a carbamide peroxide-based bleaching material significantly affected the microhardness values of filled sealant materials. The bleaching agent did not affect the microhardness of the unfilled sealant. CLINICAL SIGNIFICANCE: The results of this in vitro study suggest that the bleaching agents altered the surface hardness of filled sealant restorative materials. This could possibly lead to increased wear and surface roughness. © 2006, Copyright the authors.

Cardiovascular responses produced by central injection of hydrogen peroxide in conscious rats

Reactive oxygen species (ROS) have been shown to modulate neuronal synaptic transmission and may play a role on the autonomic control of the cardiovascular system. In this study we investigated the effects produced by hydrogen peroxide (H 2O 2) injected alone or combined with the anti-oxidant agent N-acetil-l-cysteine (NAC) or catalase into the fourth brain ventricle (4th V) on mean arterial pressure and heart rate of conscious rats. Moreover the involvement of the autonomic nervous system on the cardiovascular responses to H 2O 2 into the 4th V was also investigated. Male Holtzman rats (280-320 g) with a stainless steel cannula implanted into the 4th V and polyethylene cannulas inserted into the femoral artery and vein were used. Injections of H 2O 2 (0.5, 1.0 and 1.5 μmol/0.2 μL, n = 6) into the 4th V produced transient (for 10 min) dose-dependent pressor responses. The 1.0 and 1.5 μmol doses of H 2O 2 also produced a long lasting bradycardia (at least 24 h with the high dose of H 2O 2). Prior injection of N-acetyl-l-cysteine (250 nmol/1 μL/rat) into the 4th V blockade the pressor response and attenuated the bradycardic response to H 2O 2 (1 μmol/0.5 μL/rat, n = 7) into the 4th V. Intravenous (i.v.) atropine methyl bromide (1.0 mg/kg, n = 11) abolished the bradycardia but did not affect the pressor response to H 2O 2. Prazosin hydrochloride (1.0 mg/kg, n = 6) i.v. abolished the pressor response but did not affect the bradycardia. The increase in the catalase activity (500 UEA/1 μL/rat injected into the 4th V) also abolished both, pressor and bradycardic responses to H 2O 2. The results suggest that increased ROS availability into 4th V simultaneously activate sympathetic and parasympathetic outflow inducing pressor and bradycardic responses. © 2006 Elsevier Inc. All rights reserved.

A multicommuted flow-based system for hydrogen peroxide determination by chemiluminescence detection using a photodiode

A simple, rapid, and automated assay for hydrogen peroxide in pharmaceutical samples was developed by combining the multicommutation system with a chemiluminescence (CL) detector. The detection was performed using a spiral flow-cell reactor made from polyethylene tubing that was positioned in front of a photodiode. It allows the rapid mixing of CL reagent and analyte and simultaneous detection of the emitted light. The chemiluminescence was based on the reaction of luminol with hydrogen peroxide catalyzed by hexacyanoferrate(III). The feasibility of the flow system was ascertained by analyzing a set of pharmaceutical samples. A linear response within the range of 2.2-210 μmol l-1 H2O2 with a LD of 1.8 μmol l-1 H2O2 and coefficient of variations smaller than 0.8% for 1.0×10-5 mol l-1 and 6.8×10-5 mol l-1 hydrogen peroxide solutions (n=10) were obtained. Reagents consumption of 90 μg of luminol and 0.7 mg of hexacyanoferrate(III) per determination and sampling rate of 200 samples per hour were also achieved. Copyright © Taylor & Francis Group, LLC.

A simple spectrophotometric method for the determination of methyldopa using p-chloranil in the presence of Hydrogen Peroxide

A simple, rapid and sensitive spectrophotometric method has been developed for the determination of methyldopa in pharmaceutical formulations. The method is based on the reaction between tetrachloro-p-benzoquinone (p-chloranil) and methyldopa, accelerated by hydrogen peroxide (H 2O 2), producing a violet-red compound (λmax = 535 nm) at ambient temperature (25.0 ± 0.2°C). Experimental design methodologies were used to optimize the measurement conditions. Beer's law is obeyed in a concentration range from 2.10 × 10 -4 to 2.48 × 10 -3 mol L -1 (r = 0.9997). The limit of detection was 7.55 × 10 -6 mol L -1 and the limit of quantification was 2.52 × 10 -5 mol L -1. The intraday precision and interday precision were studied for 10 replicate analyses of 1.59 × 10 -3 mol L -1 methyldopa solution and the respective coefficients of variation were 0.7 and 1.1%. The proposed method was successfully applied to the determination of methyldopa in commercial brands of pharmaceuticals. No interferences were observed from the common excipients in the formulations. The results obtained by the proposed method were favorably compared with those given by the Brazilian Pharmacopoeia procedure at 95% confidence level.

Immunostimulatory effects of the phenolic compounds from lichens on nitric oxide and hydrogen peroxide production

The effects of isolated compounds from Brazilian lichens and their derivatives on H 2O 2 and NO production were studied using murine macrophages as a part of an attempt to understand their possible immunomodulatory properties. The compound cytotoxicity was studied using MTT assay. Macrophage stimulation was evaluated by the determination of NO (Griess assay) and H 2O 2 (horseradish peroxidase/phenol red) in supernatants of peritoneal macrophage cultures of Swiss mice. This research demonstrated stimulatory activities of some phenolic compounds isolated from lichens and their derivatives on H 2O 2 and NO production. Structure-activity relationships suggest several synthetic directions for further improvement of immunological activity.

Lichen metabolites modulate hydrogen peroxide and nitric oxide in mouse macrophages

The activities of perlatolic acid (1), atranorin (2), and lecanoric acid (3) and their derivatives, such as orsellinates and β-methyl orsellinates obtained by alcoholysis, were assessed for stimulation of the release of hydrogen peroxide and nitric oxide in cultures of peritoneal macrophage cells from mice. The hydrogen peroxide production was estimated by oxidation of phenol red, while the Griess reagent was used to determine the nitric oxide production. 1 and 4-methoxy-ethyl orsellinate (XVII) were the compounds that induced the greatest release of H 2O 2, whereas n-pentyl orsellinate (IV), iso-propyl orsellinate (V), sec-butyl orsellinate (VI), and XVII induced a small release of NO. These results indicate that lichen products and their derivatives have potential immune-modulating activities. © 2009 Verlag der Zeitschrift für Naturforschung, Tübingen.

Indirect cytotoxicity of a 35% hydrogen peroxide bleaching gel on cultured odontoblast-like cells

The aim of this study was to evaluate the trans-enamel and trans-dentinal effects of a 35% hydrogen peroxide (H2O2) bleaching gel on odontoblast-like cells. Enamel/dentin discs obtained from bovine incisors were mounted in artificial pulp chambers (APCs). Three groups were formed: G1- 35% H2O2; G2- 35% H2O2 + halogen light application; G3- control. The treatments were repeated 5 times and the APCs were incubated for 12 h. Then, the extract was collected and applied for 24 h on the cells. Cell metabolism, total protein dosage and cell morphology were evaluated. Cell metabolism decreased by 62.09% and 61.83% in G1 and G2, respectively. The depression of cell metabolism was statistically significant when G1 and G2 were compared to G3. Total protein dosage decreased by 93.13% and 91.80% in G1 and G2, respectively. The cells in G1 and G2 exhibited significant morphological alterations after contact with the extracts. Regardless of halogen light application, the extracts caused significantly more intense cytopathic effects compared to the control group. After 5 consecutive applications of a 35% H2O2 bleaching agent, either catalyzed or not by halogen light, products of gel degradation were capable to diffuse through enamel and dentin causing toxic effects to the cells.

An in vitro thermal analysis during different light-activated hydrogen peroxide bleaching

This study measured the critical temperature reaching time and also the variation of temperature in the surface of the cervical region and within the pulp chamber of human teeth submitted to dental bleaching using 35% hydrogen peroxide gel activated by three different light sources. The samples were randomly divided into 3 groups (n = 15), according to the catalyst light source: Halogen Light (HL), High Intensity Diode Laser (DL), and Light Emmited Diode (LED). The results of temperature variation were submitted to the analysis of variance and Tukey test with p < 0.05. The temperature increase (mean value and standard deviation) inside the pulp chamber for the HL group was 6.8 ± 2.8°C; for the DL group was 15.3 ± 8.8°C; and for the LED group was 1.9 ± 1.0°C for. The temperature variation (mean value and standard deviation) on the tooth surface, for the group irradiated with HL was 9.1 ± 2.2°C; for the group irradiated with DL were 25.7 ± 18.9°C; and for the group irradiated with LED were 2.6 ± 1.4°C. The mean temperature increase values were significantly higher for the group irradiated with DL when compared with groups irradiated with HL and LED (p < 0.05). When applying the inferior limits of the interval of confidence of 95%, an application time of 38.7 s was found for HL group, and 4.4 s for DL group. The LED group did not achieve the critical temperatures for pulp or the periodontal, even when irradiated for 360 s. The HL and DL light sources may be used for dental bleaching for a short period of time. The LED source did not heat the target tissues significantly within the parameters used in this study. © 2010 Pleiades Publishing, Ltd.

Effects of five carbamide peroxide bleaching gels on composite resin microhardness.

The purpose of this study was to evaluate the effects of five home bleaching products containing 15-16% carbamide peroxide on the microhardness of microhybrid composite resin Z-250 (3M/Espe). A total of 72 specimens were fabricated in cylindrical acrylic matrices (4 x 2 mm), filled with composite resin and photo-activated for 40 seconds. They were divided in 6 study groups (n = 12), according to the bleaching product: Review (SS White), Magic Bleaching (Vigodent), Opalescence (Ultradent), Whiteness Perfect (FGM), Claridex (Biodinâmica), and a control group (not bleached). Specimens were exposed to 1 cc of bleaching gel for 6 hours daily for 2 weeks. The control group specimens were kept in artificial saliva throughout this time. All the specimens were then analyzed in a microhardness tester. Knoop hardness measurements were performed, and the results were submitted to parametric statistical analysis (analysis of variance and Tukey's test). Mean Knoop values and standard deviation were: baseline, 68.52a (4.28); control, 63.42b (7.16); Whiteness Perfect, 57.57c (1.81); Magic Bleaching, 57.22c (3.84); Opalescence, 57.03cd (4.00); Claridex, 53.64de (3.33); Review 51.45e (2.82). Identical letters mean statistical equality according to Tukey's test at the 5% significance level. The products significantly decreased Z-250 (3M/Espe) microhardness.