Innate Immune Response against Staphylococcus aureus Preincubated with Subinhibitory Concentration of trans-Anethole.

The study aimed to analyze morphological and functional changes of Staphylococcus aureus cells due to trans-anethole (a terpenoid and the major constituent of fennel, anise, or star anise essential oils) exposition, and their consequences for human neutrophils phagocytic activity as well as IL-8 production (recognized as the major chemoattractant). The investigation included the evaluation of changes occurring in S. aureus cultures, i.e., staphyloxanthin production, antioxidant activities, cell size distribution, and cells composition as a result of incubation with trans-anethole. It was found that the presence of trans-anethole in the culture medium reduced the level of staphyloxanthin production, as well as decreased antioxidant activities. Furthermore, trans-anethole-treated cells were characterized by larger size and a tendency to diffuse in comparison to the non-treated cells. Several cell components, such as phospholipids and peptidoglycan, were found remarkably elevated in the cultures treated with trans-anethole. As a result of the aforementioned cellular changes, the bacteria were phagocytized by neutrophils more efficiently (ingestion and parameters associated with killing activity were at a higher level as compared to the control system). Additionally, IL-8 production was at a higher level for trans-anethole modified bacteria. Our results suggest that trans-anethole represents a promising measure in combating severe staphylococcal infections, which has an important translational potential for clinical applications.

Influence of macerated fenugreek (Trigonella foenum graecum) oil added to trout feed at the different rates on the Feed Conversion Rate (FCR), body length, blood parameters and Nitroblue Tetrazolium (NBT) values ​​of rainbow trout (Oncorhynchus mykiss Wal.

In this study, 1% and 2% of macerated fenugreek oil was added to the feeds of rainbow trout with an average weight of 25.79 ± 1.5 g. At the end of the study, growth rate, blood parameters and NBT (Nitroblue Tetrazolium) level of rainbow trout were determined. The best feed ratio (FCR) was observed in the control group (0.77). Statistically significant differences were found only in MID values (P<0.05), although there was a numerical increase in all blood parameters. There was no statistically significant difference between NBT levels (P> 0.05). Although the best weight gain was in the control group as in the FCR values, the maximum elongation was measured at D1 and then at D2 (P <0.05). The best survival rate was obtained with 96.66% in D1 while the worst was observed in D2 with 60% (p<0,05).

Inhibitory Effects on NO Production and DPPH Radicals and NBT Superoxide Activities of Diarylheptanoid Isolated from Enzymatically Hydrolyzed Ehthanolic Extract of Alnus sibirica.

Alnus sibirica (AS) is geographically distributed in Korea, Japan, Northeast China, and Russia. Various anti-oxidant, anti-inflammation, anti-atopic dermatitis and anti-cancer biological effects of AS have been reported. Enzymatic hydrolysis decomposes the sugar bond attached to glycoside into aglycone which, generally, has a superior biological activity, compared to glycoside. Enzymatic hydrolysis of the extract (EAS) from AS was processed and the isolated compounds were investigated-hirsutanonol (1), hirsutenone (2), rubranol (3), and muricarpon B (4). The structures of these compounds were elucidated, and the biological activities were assessed. The ability of EAS and the compounds (1-4) to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and Nitroblue tetrazolium (NBT) superoxide, and to inhibit NO production was evaluated in vitro. EAS showed more potent antioxidant and anti-inflammatory activity than AS. All investigated compounds showed excellent antioxidant and anti-inflammatory activities.

The effects of common yarrow (Achillea millefolium Linnaeus), cinnamon (Cinnamomum zeylanicum Blume) and rosemary (Rosemarinus officinalis Linnaeus) hydrosols on the some immunological and hematological parameters of common carp (Cyprinus carpio L., 1758).

In this study, the effects of some plant hydrosols (distilled plant waters) based upon some hematological parameters and Nitroblue Tetrazolium (NBT) activities in the common carp (Cyprinus carpio Linnaeus, 1758) infected with Yersinia ruckeri were investigated. In the trial, it was utilized totally 200 common carps with 54.3±6.7 g mean live weight and 15.7±1.8 cm mean total lenght. The 10% rate of the common yarrow (Achillea millefolium Linnaeus) hydrosol; 0.5% rate of the cinnamon (Cinnamomum zeylanicum Blume) hydrosol; and 5% rate of the rosemary (Rosemarinus officinalis Linnaeus) hydrosol were applied to fish as a bath treatment. The erythrocyte (RBC), leukocyte count (WBC), hematocrit value (HCT), haemoglobin amount (Hg), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and activities of NBT in the blood samples taken from the caudal vena of the control and experimental fish groups were analyzed in the 7th, 14th, and 21st days of the exposure treatment. At the end of the research, HCT, Hg, RBC, WBC, MCH and MCV values decreased in the C-2 Group (the control group contain pathogen) compared to the C-1 Group (the control group no contain pathogen), except MCHC value. The NBT activities in the C-1 Groups increased at the 14th day, but decreased quite a few at the 21st day. It has been consequently reached the conclusion that the bath treatments of the some plant hydrosols might be beneficial in increasing of antibacterial properties and in strengthening of defense mechanisms of common carp against Y. ruckeri pathogen.

Multi-centre assessment of nitroblue tetrazolium reactivity in human semen as a potential marker of oxidative stress.

The nitroblue tetrazolium (NBT) reaction as a tracer of oxidative stress was examined in 707 ejaculates from seven clinics. Semen was initially surveyed by classifying the NBT reaction using a pre-established rank for the Oxisperm® test based on three colourimetric levels: L1, low (n = 141 [20%]); L2, medium (n = 538 [76%]) and L3, high (n = 28 [4%]). L3 was indicative of a high level of superoxide anions. Halosperm® chromatin dispersion assay was used to analyse samples of ejaculates 30 min after ejaculation; no difference was found in DNA fragmentation of L1 or L3; L3 category semen samples incubated for 24 h at 37oC showed a significantly faster rate (P < 0.001) of DNA damage than those in L1. The NBT reaction was further characterized in the ejaculates of 100 patients to determine the relative contribution of seminal plasma, spermatozoa, or both. Seminal plasma was the most significant fraction of •O2- localization, whereas sperm fractions generated detectable reactive oxygen species in only 32% of the ejaculates. Formazan precipitates were primarily associated with the sperm mid-piece and seminal leukocytes; however, not all spermatozoa stained positive to formazan and not all leukocytes presented with equivalent production of superoxide anions.

Proteomic Analysis Reveals the Dynamic Role of Silicon in Alleviation of Hyperhydricity in Carnation Grown In Vitro.

The present study depicted the role of silicon in limiting the hyperhydricity in shoot cultures of carnation through proteomic analysis. Four-week-old healthy shoot cultures of carnation "Purple Beauty" were sub-cultured on Murashige and Skoog medium followed with four treatments, viz. control (-Si/-Hyperhydricity), hyperhydric with no silicon treatment (-Si/+Hyperhydricity), hyperhydric with silicon treatment (+Si/+Hyperhydricity), and only silicon treated with no hyperhydricity (+Si/-Hyperhydricity). Comparing to control morphological features of hyperhydric carnations showed significantly fragile, bushy and lustrous leaf nature, while Si supply restored these effects. Proteomic investigation revealed that approximately seventy protein spots were differentially expressed under Si and/or hyperhydric treatments and were either up- or downregulated in abundance depending on their functions. Most of the identified protein spots were related to stress responses, photosynthesis, and signal transduction. Proteomic results were further confirmed through immunoblots by selecting specific proteins such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), PsaA, and PsbA. Moreover, protein-protein interaction was also performed on differentially expressed protein spots using specific bioinformatic tools. In addition, stress markers were analyzed by histochemical localization of hydrogen peroxide (H2O2) and singlet oxygen (O21-). In addition, the ultrastructure of chloroplasts in hyperhydric leaves significantly resulted in inefficiency of thylakoid lamella with the loss of grana but were recovered in silicon supplemented leaves. The proteomic study together with physiological analysis indicated that Si has a substantial role in upholding the hyperhydricity in in vitro grown carnation shoot cultures.

Chlorovirus PBCV-1 encodes an active copper-zinc superoxide dismutase.

UNLABELLED: Superoxide dismutases (SODs) are metalloproteins that protect organisms from toxic reactive oxygen species by catalyzing the conversion of superoxide anion to hydrogen peroxide and molecular oxygen. Chlorovirus PBCV-1 encodes a 187-amino-acid protein that resembles a Cu-Zn SOD with all of the conserved amino acid residues for binding copper and zinc (named cvSOD). cvSOD has an internal Met that results in a 165-amino-acid protein (named tcvSOD). Both cvSOD and tcvSOD recombinant proteins inhibited nitroblue tetrazolium reduction of superoxide anion generated in a xanthine-xanthine oxidase system in solution. tcvSOD was chosen for further characterization because it was easier to produce. Recombinant tcvSOD also inhibited a riboflavin photochemical reduction system in a polyacrylamide gel assay, which was blocked by the Cu-Zn SOD inhibitor cyanide but not by azide, which inhibits Fe and Mn SODs. A k(cat)/K(m) value for cvSOD was determined by stop-flow spectrophotometry as 1.28 × 10(8) M(-1) s(-1), suggesting that cvSOD-catalyzed O2 (-) dismutation was not a diffusion controlled encounter. The cvsod gene was expressed as a late gene, and cvSOD activity was detected in purified virions. Superoxide accumulated rapidly during virus infection, and circumstantial evidence indicates that cvSOD aids its decomposition to benefit virus replication. Cu-Zn SOD homologs have been described to occur in 3 other families of large DNA viruses, poxviruses, baculoviruses, and mimiviruses, which group as a clade. Interestingly, cvSOD does not group in the same clade as the other virus SODs but instead groups in an expanded clade that includes Cu-Zn SODs from many cellular organisms. IMPORTANCE: Virus infection often leads to an increase in toxic reactive oxygen species in the host, which can be detrimental to virus replication. Viruses have developed various ways to overcome this barrier. As reported in this article, the chloroviruses often encode and package a functional Cu-Zn superoxide dismutase in the virion that presumably lowers the concentration of reactive oxygen induced early during virus infection.

Novel, oxygen-insensitive group 5 [NiFe]-hydrogenase in Ralstonia eutropha.

Recently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H2. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium Ralstonia eutropha H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the in vivo H2 uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H2. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 µmol of H2 per min and mg of protein. However, an unexpectedly high Michaelis constant (Km) for H2 of 3.6 ± 0.5 µM was determined, which is in contrast to the previously proposed low Km of group 5 hydrogenases and makes atmospheric H2 uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H2 oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O2.

Comparative study of ß-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.).

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by ß-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by ß-glucans.

Streptococcous parauberis infection in starry flounder, Platichthys stellatus: characterization of innate immune responses following experimental infection.

Streptococcus parauberis causing systemic infections has been recognized as a major bacterial disease in olive flounder Paralichthys olivaceus in South Korea. Although an emerging outbreak of S. parauberis has affected heavily farmed fish species starry flounder Platichthys stellatus, no study of the innate immune responses and pathogenic mechanisms in starry flounder is available. In the present study, starry flounder were intraperitoneally challenged with four S. parauberis strains to investigate changes in innate immune responses. Significant increases in serum lysozyme activities, superoxide production of kidney leucocytes, and serum superoxide dismutase activities were observed following experimental injection of S. parauberis. All these data suggested that the innate immune parameters were highly modulated during the S. parauberis infection process to render protection to the starry flounder. However, S. parauberis also exhibited the mechanisms to complete disease establishment by avoiding host immune responses. S. parauberis could survive and proliferate in the mucus, serum and kidney leucocytes of starry flounder. In particular, the strain isolated from the starry flounder showed the higher survival ability than other originated strains in the tested host fish.

Effect of storage on the nitro blue tetrazolium reduction test in dog blood samples.

INTRODUCTION: The nitro blue tetrazolium (NBT) reduction test (NBTT) has been used for measuring the metabolic activity of phagocytes of mammals. Activated neutrophils transform NBT into formazan in the cytoplasm. The NBTT can detect the activation of neutrophils in peripheral blood and is used to assess neutrophil function in dogs. However, the NBTT is not used frequently in the clinical setting, as samples should be processed after blood collection. OBJECTIVE: The aim of this study was to evaluate the effect of storage on NBTT in dog blood samples. MATERIALS AND METHODS: Residual EDTA blood samples from 22 dogs were included of different ages, breeds, and sex. The buffy coat layer was separated from the blood and incubated with 0.1% NBT. The NBTT was performed at 0, 24, 48, and 72 h after the collection of blood. Blood samples were stored at 4°C until the tests were performed. Blood smears were evaluated by light microscopy, and the NBT reduction rate was reported, which represents the percentage of activated neutrophils. The NBT reduction rate was calculated after counting 300 neutrophils in each slide. RESULTS: The means of NBTT in dog blood samples at 0, 24, 48, and 72 h were 8.3%, 8.5%, 8.7%, and 7.8%, respectively. No significant differences were observed between time points. CONCLUSIONS: This study showed that the NBTT can be performed up to 72 h after the collection of canine blood if correctly refrigerated at 4°C. This finding supports the performance of the NBTT in the clinical setting.

Zymographic differentiation of [NiFe]-hydrogenases 1, 2 and 3 of Escherichia coli K-12.

BACKGROUND: When grown under anaerobic conditions, Escherichia coli K-12 is able to synthesize three active [NiFe]-hydrogenases (Hyd1-3). Two of these hydrogenases are respiratory enzymes catalysing hydrogen oxidation, whereby Hyd-1 is oxygen-tolerant and Hyd-2 is considered a standard oxygen-sensitive hydrogenase. Hyd-3, together with formate dehydrogenase H (Fdh-H), forms the formate hydrogenlyase (FHL) complex, which is responsible for H2 evolution by intact cells. Hydrogen oxidation activity can be assayed for all three hydrogenases using benzyl viologen (BV; Eo' = -360 mV) as an artificial electron acceptor; however ascribing activities to specific isoenzymes is not trivial. Previously, an in-gel assay could differentiate Hyd-1 and Hyd-2, while Hyd-3 had long been considered too unstable to be visualized on such native gels. This study identifies conditions allowing differentiation of all three enzymes using simple in-gel zymographic assays. RESULTS: Using a modified in-gel assay hydrogen-dependent BV reduction catalyzed by Hyd-3 has been described for the first time. High hydrogen concentrations facilitated visualization of Hyd-3 activity. The activity was membrane-associated and although not essential for visualization of Hyd-3, the activity was maximal in the presence of a functional Fdh-H enzyme. Furthermore, through the use of nitroblue tetrazolium (NBT; Eo' = -80 mV) it was demonstrated that Hyd-1 reduces this redox dye in a hydrogen-dependent manner, while neither Hyd-2 nor Hyd-3 could couple hydrogen oxidation to NBT reduction. Hydrogen-dependent reduction of NBT was also catalysed by an oxygen-sensitive variant of Hyd-1 that had a supernumerary cysteine residue at position 19 of the small subunit substituted for glycine. This finding suggests that tolerance toward oxygen is not the main determinant that governs electron donation to more redox-positive electron acceptors such as NBT. CONCLUSIONS: The utilization of particular electron acceptors at different hydrogen concentrations and redox potentials correlates with the known physiological functions of the respective hydrogenase. The ability to rapidly distinguish between oxygen-tolerant and standard [NiFe]-hydrogenases provides a facile new screen for the discovery of novel enzymes. A reliable assay for Hyd-3 will reinvigorate studies on the characterisation of the hydrogen-evolving FHL complex.

High-throughput analysis of gene expression on tissue sections by in situ hybridization.

Genome-scale sequencing projects, high-throughput RNAi screens, systematic gene targeting, and system-biology-based network predictions all depend on a validation of biological significance in order to understand the relevance of a particular finding. Such validation, for the most part, rests on low-throughput technologies. This article provides protocols that, in combination with suitable instrumentation, make possible a semi-automated analysis of gene expression on tissue sections by means of in situ hybridization. Knowledge of gene expression localization has the potential to aid, and thereby accelerate, the validation of gene functions.

Functional state analysis of phagocytic cells of patients with type 2 diabetes and pulmonary tuberculosis.

BACKGROUND: The phagocytic function in pulmonary tuberculosis (PTB) and Type 2 diabetes (T2D) has been explored mainly in macrophages but not in polymorphonuclears (PMN). The purpose of this study was to determine the functional status of PMN leukocytes in patients with pulmonary tuberculosis (PTB), Type 2 diabetes (T2D), and in patients with both diseases. METHODS: An observational, prospective, and comparative study was carried out. 30 ambulatory patients with T2D, 10 with PTB undergoing treatment and 10 patients with PTB and T2D, and 44 healthy subjects were studied. PMN leukocytes were separated, the capacity of these cells to produce hydrogen peroxide and to reduce nitroblue tetrazolium (NBT) in response to stimulus with the phorbolic ester of myristic acid (PMA) was measured; and the capacity of PMN leukocytes to adhere to surfaces was determined. RESULTS: Concerning the test for adherence, on comparing healthy subjects with patients with T2D+PTB, we observed a clear decrease in cellular adherence in the group of patients with both diseases; it was statistically significant (p = 0.007).With regard to phagocytic function, we observed that in NBT reduction as well as in hydrogen peroxide production, statistically significant differences were not obtained on comparing healthy subjects with any of the three groups of patients. CONCLUSIONS: We observed a clear decrease in cellular adherence when both diseases co-exist. These results could indicate the need for the co-existence of T2D and TB to cause deterioration in the cells' adherence activity. The microtechniques employed permit the evaluation in a practical manner of certain phagocytic-activity expressions.

A mutation of OSOTP 51 leads to impairment of photosystem I complex assembly and serious photo-damage in rice.

Gene expression in chloroplasts is regulated by many nuclear-encoded proteins. In this study, we isolated a rice (Oryza sativa subsp. japonica) mutant osotp51 with significant reduction in photosystem I (PSI). The osotp51 is extremely sensitive to light and accumulates a higher level of reactive oxygen species. Its leaves are almost albino when grown at 40 µmol photons/m(2) per s. However, grown at 4 µmol photons/m(2) per s, osotp51 has a similar phenotype to the wild-type. 77K chlorophyll fluorescence analysis showed a blue shift in the highest peak emission from PSI in osotp51. In addition, the level of PSI and PSII dimer is dramatically reduced in osotp51. OSOTP 51 encodes a pentatricopeptide repeats protein, homologous to organelle transcript processing 51 in Arabidopsis. Loss-of-function OSOTP51 affects intron splicing of a number of plastid genes, particularly the ycf3 coding a protein involved in the assembly of PSI complex. OSOTP51 is functionally conserved in higher plants. The mutation of osotp51 indirectly leads to a widespread change in the structure and functions of PSI, results in severe photoinhibition, and finally dies, even when grown under very low light intensity.

Enhanced colorimetric detection on porous microarrays using in situ substrate production.

A new technique is reported for the enhanced colorimetric detection of multiplexed hybridization onto porous membrane-based microarrays. This approach combines the use of horseradish peroxidase (HRP) as a label together with a chromogen substrate and a local production of the hydrogen peroxide required for substrate oxidation. This in situ production of coreagent is obtained using glucose oxidase (GOx) directly immobilized within the microarray porous membrane mesh. The oxidation of glucose by the immobilized GOx produces hydrogen peroxide which itself enables the oxidation of TMB (3,3',5,5'-tetramethylbenzidine) by HRP and yields a blue precipitate on positive spots. Thanks to a coreagent overconcentration within the membrane, this design drastically surpasses the performances of the standard TMB/H(2)O(2) kit used for peroxidase label detection. The obtained target limit of detection is then 50 times lower (20 pM) than the one obtained with the standard kit approach, and the dynamic range expands at least one decade. Furthermore, the developed method was shown to compete well with the widely used alkaline phosphatase-BCIP (5-bromo-4-chloro-3-indolyl phosphate)/NBT (nitro blue tetrazolium chloride) readout while minimizing background signal. The method was finally successfully applied to the multiplex detection of single nucleotide polymorphisms (SNPs) in complex PCR samples. The background lowering was impacted here positively on the SNPs' detection by increasing the complementary/noncomplementary signal ratio.

In vivo effect of three fractions of Larrea divaricata Cav. (jarilla) on the innate immune system: macrophage response against Candida albicans.

Larrea divaricata Cav. (jarilla) is a plant with well-documented applications in folk medicine in Argentina. In this study, we aimed to evaluate functional parameters of peritoneal macrophages isolated from mice injected with three fractions (F1, F2 and F3) of L. divaricata. The response of macrophages against Candida albicans was evaluated. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, apoptosis was evaluated using Giemsa, acridine orange/ethidium bromide and ladder assay, oxidative burst was assayed using nitroblue tetrazolium test and nitrite production using Griess assay. Cell stimulation and their ability to kill C. albicans in vitro were measured. The number and cell viability were similar to controls. However, we found that F1 induces pre-activation of macrophages, and this pre-activation is enhanced by C. albicans. The effects exerted by F1 make it more important than F2 and F3 for the treatment of disseminated candidiasis in patients with immunodeficiency diseases such as AIDS and chronic granulomatous disease, among others.

Failure to trigger the oxidative metabolic burst by normal macrophages: possible mechanism for survival of intracellular pathogens.

As previously reported, normal human monocytes (11) and activated mouse macrophages (9) are able to kill or inhibit intracellular replication of Toxoplasma that are not antibody coated, whereas normal human and mouse macrophages are not (7, 9). Each of these types of mononuclear phagocytes is able to kill antibody-coated Toxoplasma. In our studies, phagocytosis of antibody-coated Toxoplasma stimulated the respiratory burst by each of these types of mononuclear phagocytes, whereas phagocytosis of organisms that were not antibody coated stimulated the respiratory burst only by human monocytes and by activated mouse macrophages. Phagocytosis of Toxoplasma did not inhibit production of reactive oxygen metabolites by normal macrophages; rather, it failed to stimulate their production. Killing of Toxoplasma by monocytes from a child with X-linked chronic granulomatous disease and his heterozygote mother was impaired. Thus, reactive oxygen metabolites, perhaps in conjunction with lysosomal contents, appear to be first-line mechanisms whereby mononuclear phagocytes kill this organism. We were not able to determine the exact mechanisms whereby mononuclear phagocytes inhibit the replication of those Toxoplasma that were not killed, although both oxygen-dependent and other nonlysosomal mechanisms may be involved. The differences we observed in oxidative response to phagocytosis of Toxoplasma appear to be one determinant of the antimicrobial activity of these cells and may account for the ability of some intracellular pathogens to survive within phagocytes. These differences may be membrane related. Further studies of Toxoplasma membranes, phagocyte membrane receptors for Toxoplasma, and membrane-related mechanisms for activation of the respiratory burst are needed to define their true basis.

Antioxidant capacity of binuclear Cu(II)-cyclophanes, insights from two synthetic bioactive molecules.

The compounds 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza-17,40-dioxa[10.1.10.1]paracyclophane and 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza[10.1.10.1]paracyclophane binuclear copper complexes (Cu2PO and Cu2PC, respectively) were studied by determining their antioxidant capacity using the TROLOX equivalent antioxidant capacity (TEAC) assay, and their cytotoxicity on cultured cells, as well as the superoxide dismutase (SOD)-like activity. Cu2PO had an antioxidant capacity (0.1 g eq TROLOX mol−1) within the order of magnitude of ascorbic acid, and both, Cu2PO and Cu2PC were nontoxic to cultured peripheral mononuclear blood cells. The SOD-like activity was evaluated using the nitroblue tetrazolium assay, and both compounds presented an excellent activity: for Cu2PO, the IC50 was 52 nM and for Cu2PC an IC50 of 0.5 µM was obtained comparable to CuZn SOD IC50 17 nM (Fernandes et al., J Inorg Biochem 2007;101:849­858). These results suggest that synthetic binuclear macrocycles are good candidates to be used as synthetic bioactive molecules with applications in biomedicine.

Engineering of glycerol dehydrogenase for improved activity towards 1, 3-butanediol.

The objective of this study was to use protein engineering techniques to enhance the catalytic activity of glycerol dehydrogenase (GlyDH) on racemic 1, 3-butanediol (1, 3-BDO) for the bioproduction of the important pharmaceutical intermediate 4-hydroxy-2-butanone. Three GlyDH genes (gldA) from Escherichia coli K-12, Salmonella enterica, and Klebsiella pneumoniae MGH78578 were shuffled to generate a random mutagenesis library. The nitroblue tetrazolium/phenazine methosulfate high throughput screening protocol was used to select four chimeric enzymes with up to a 2.6-fold improved activity towards 1, 3-BDO. A rational design method was also employed to further improve the enzyme activity after DNA shuffling. Based on the homology model of GlyDH (Escherichia coli), Asp121 was predicted to influence 1, 3-BDO binding and replaced with Ala by site-directed mutagenesis. Combination of the mutations from both DNA shuffling and rational design produced the best mutant with a V (max) value of 126.6 U/mg, a 26-fold activity increase compared with that of the wild type GlyDH from E. coli.