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.

NADPH diaphorase detects S-nitrosylated proteins in aldehyde-treated biological tissues.

NADPH diaphorase is used as a histochemical marker of nitric oxide synthase (NOS) in aldehyde-treated tissues. It is thought that the catalytic activity of NOS promotes NADPH-dependent reduction of nitro-blue tetrazolium (NBT) to diformazan. However, it has been argued that a proteinaceous factor other than NOS is responsible for producing diformazan in aldehyde-treated tissues. We propose this is a NO-containing factor such as an S-nitrosothiol and/or a dinitrosyl-iron (II) cysteine complex or nitrosated proteins including NOS. We now report that (1) S-nitrosothiols covalently modify both NBT and TNBT, but only change the reduction potential of NBT after modification, (2) addition of S-nitrosothiols or ß- or α-NADPH to solutions of NBT did not elicit diformazan, (3) addition of S-nitrosothiols to solutions of NBT plus ß- or α-NADPH elicited rapid formation of diformazan in the absence or presence of paraformaldehyde, (4) addition of S-nitrosothiols to solutions of NBT plus ß- or α-NADP did not produce diformazan, (5) S-nitrosothiols did not promote NADPH-dependent reduction of tetra-nitro-blue tetrazolium (TNBT) in which all four phenolic rings are nitrated, (6) cytoplasmic vesicles in vascular endothelial cells known to stain for NADPH diaphorase were rich in S-nitrosothiols, and (7) procedures that accelerate decomposition of S-nitrosothiols, markedly reduced NADPH diaphorase staining in tissue sections subsequently subjected to paraformaldehyde fixation. Our results suggest that NADPH diaphorase in aldehyde-fixed tissues is not enzymatic but is due to the presence of NO-containing factors (free SNOs or nitrosated proteins such as NOS), which promote NADPH-dependent reduction of NBT to diformazan.

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.

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.

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).

Induction of apoptosis and erythroid differentiation of human chronic myelogenous leukemia K562 cells by low concentrations of lidamycin.

Apoptosis induction and differentiation of promyelocytic leukemic cells into mature cells are major strategies for the drug-based treatment of leukemia. Lidamycin (LDM) which is a member of the enediyne antibiotic family exhibits extreme cytotoxicity. In the present study, the induction of apoptosis and differentiation in human chronic myeloid leukemia K562 cells by low concentrations of lidamycin were investigated. K562 cells were treated with lidamycin at various concentrations for 48 h, and accumulated in the metaphase as determined in previous experiments. Cell viability was determined using a Cell Counting Kit-8 (CCK-8) assay and the IC50 value of lidamycin was 0.1±3.2 nM. Induction of apoptosis was investigated morphologically by acridine orange/ethidium bromide (AO/EtBr) staining. Growth inhibition and apoptosis induction were observed in cells treated with low concentrations of lidamycin. In addition, western blot analysis revealed that treatment of the K562 cells with lidamycin at low concentrations upregulated the expression of caspase-8 and caspase-3. The induction of differentiation in human chronic myeloid leukemia K562 cells by lidamycin at low concentrations was also investigated. The nitroblue tetrazolium reduction ability of K562 cells was increased following treatment with lidamycin. Low concentrations of lidamycin triggered erythroid differentiation among K562 cells, indicated by morphological changes, increased hemoglobin content, and the expression of cell surface antigens such as CD71. Additionally the expression of GATA-binding factor 1 (GATA-1) protein in low concentration lidamycin-treated K562 cells was increased. The results of the present study suggest that a low-concentration lidamycin exerts effects on apoptosis and erythroid differentiation induction by increasing the expression of caspases and GATA-1 protein. Lidamycin may serve a positive role in relevant targeted chemotherapy and may represent a potential candidate for chronic myelogenous leukemia differentiation-inducing treatment.

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.

Chemical Composition and Antioxidant, Analgesic, and Anti-Inflammatory Effects of Methanolic Extract of Euphorbia retusa in Mice.

Plants provide an alternative source to manage different human disorders due to various metabolites. The aim of this study is to investigate the phytochemical constituents of the methanolic extracts of Euphorbia retusa and to evaluate their antioxidant, anti-inflammatory, and analgesic activities. The phytochemical results obtained by HPLC and by chemical assay reactions have revealed the richness of the methanolic extract of E. retusa in active compounds, in particular polyphenols, flavonoids, and tannins. The methanolic extract shows significant antioxidant activities in vitro, in the DPPH and the FRAP assays. The antinociceptive activity was evaluated using acetic acid and hot-plate models of pain in mice. The anti-inflammatory activity was evaluated by carrageenan-induced paw edema. Oral pretreatment with the methanolic extract of E. retusa (200 mg/kg) exhibited a significant inhibition of pain induced either by acetic acid or by the heating plate and in a manner comparable to the standard drug paracetamol. E. retusa significantly reduced paw edema starting from the 3rd hour after carrageenan administration by increasing the activity of antioxidant enzymes (SOD, CAT, and GPx) in liver and paw tissues and decreasing the levels of MDA. These results may confirm the interesting potential of this plant as a treatment of various inflammatory and pain diseases.

Early axis growth during seed germination is gravitropic and mediated by ROS and calcium.

In plant establishment, seed germination is characterized by the emergence of a radicle for secured anchorage to the soil and nutrient and water uptake. Early growth of germinating axes appears to be gravisensitive, and the regulation of this process is largely uncharacterized, particularly in case of epigeally germinating species. Our previous work on the germination of Vigna radiata seeds demonstrated the role of apoplastic reactive oxygen species (ROS) in germination-associated axis growth. This study attempts to explore a possibly similar role of ROS in the gravitropic bending of germinating axes. Pharmacological and histological studies correlated the curvature growth of the axis (due to cell elongation in the cortical region of the upper side) with apoplastic superoxide accumulation. The superoxide was produced by diphenylene iodonium chloride (DPI)-insensitive NADH oxidase, which was different from the DPI-sensitive NADPH oxidase active in the apical elongation zone of the radicle. This NADH oxidase was differentially controlled by IAA, and its activation required influx of apoplastic Ca2+. This study shows that the early axis growth in germinating seeds is gravisensitive, which is distinct spatially as well as temporally from the elongation growth of the axis (radicle) and controlled by auxin and cytosolic Ca2+ through NADH oxidase-dependent ROS production.

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.

A paper/polymer hybrid microfluidic microplate for rapid quantitative detection of multiple disease biomarkers.

Enzyme linked immunosorbent assay (ELISA) is one of the most widely used laboratory disease diagnosis methods. However, performing ELISA in low-resource settings is limited by long incubation time, large volumes of precious reagents, and well-equipped laboratories. Herein, we developed a simple, miniaturized paper/PMMA (poly(methyl methacrylate)) hybrid microfluidic microplate for low-cost, high throughput, and point-of-care (POC) infectious disease diagnosis. The novel use of porous paper in flow-through microwells facilitates rapid antibody/antigen immobilization and efficient washing, avoiding complicated surface modifications. The top reagent delivery channels can simply transfer reagents to multiple microwells thus avoiding repeated manual pipetting and costly robots. Results of colorimetric ELISA can be observed within an hour by the naked eye. Quantitative analysis was achieved by calculating the brightness of images scanned by an office scanner. Immunoglobulin G (IgG) and Hepatitis B surface Antigen (HBsAg) were quantitatively analyzed with good reliability in human serum samples. Without using any specialized equipment, the limits of detection of 1.6 ng/mL for IgG and 1.3 ng/mL for HBsAg were achieved, which were comparable to commercial ELISA kits using specialized equipment. We envisage that this simple POC hybrid microplate can have broad applications in various bioassays, especially in resource-limited settings.

In vivo protein targets for increased quinoprotein adduct formation in aged substantia nigra.

The selective vulnerability of dopaminergic neurons in the substantia nigra pars compacta in Parkinson's disease, a late age onset neurodegenerative disorder, indicates the involvement of dopamine metabolism in the pathogenesis. Dopamine oxidation produces dopamine o-quinone, which covalently modifies cysteinyl proteins forming quinoprotein adduct. Although quinoprotein formation correlates with increased dopaminergic neurotoxicity, the in vivo protein targets for quinone modification remain unclear. Using two-dimensional gel electrophoresis and nitroblue tetrazolium/glycinate redox-cycling staining, we compared quinoprotein adducts in the substantia nigra of 2- and 15-month old rats and for the first time identified the in vivo protein targets with increased quinone modification in aged substantia nigra. Interestingly, several key enzymes in energy metabolism and mitochondrial function were selectively modified by quinone during aging. In vitro analyses confirmed that two of identified enzymes, l-lactate dehydrogenase (LDH) and malate dehydrogenase (MDH), were readily conjugated by dopamine o-quinone, resulting in a significant reduction in enzyme activity. Since the proteomic approach to detect quinoprotein adducts represents a single analysis comparing pools of substantia nigra from young or old rats, these findings need to be verified in the future. Nonetheless, our results reveal that the enzymatic activity of LDH and MDH can be compromised by quinone modification, suggesting a role of energy metabolism impairment in the selective vulnerability of aged substantia nigra dopaminergic neurons in Parkinson's disease.

Innate Cellular Immunity in Newly Diagnosed Pulmonary Tuberculosis Patients and During Chemotherapy.

BACKGROUND: Leukocyte migration (LM) and intracellular killing aspects of the innate immune response play important roles in protection against and containment and cure of Mycobacterium tuberculosis infection, and thus may be exploited as immunotherapeutic targets to improve the management and treatment outcomes of patients with tuberculosis (TB). OBJECTIVES: The aim of this study was to assess LM and mediators of intracellular killing in patients with TB at the time of diagnosis and during anti-TB chemotherapy and compare them with apparently healthy controls. METHODS: We recruited 24 patients who were newly diagnosed with pulmonary TB and 20 apparently healthy individuals. Blood was drawn from patients with TB at the time of diagnosis, and after 2, 4, and 6 months of anti-TB chemotherapy and control. In vitro percentage LM (%LM) upon stimulation with Bacillus Calmette-Guérin vaccine, percentage nitroblue tetrazolium (%NBT) reduction, plasma concentrations of hydrogen peroxide (H2O2), and nitric oxide (NO) were assessed in both groups. FINDINGS: Percentage NBT was significantly reduced in patients with TB at 2 months of anti-TB chemotherapy compared with patients at diagnosis and in healthy controls, whereas %LM was significantly increased in patients at 4 months of anti-TB chemotherapy compared with patients at diagnosis and controls. Mean plasma H2O2 and NO were significantly reduced in patients at diagnosis and throughout the period of anti-TB chemotherapy compared with the control group. Significant decreases were demonstrated in mean plasma H2O2 and NO in patients at 2 and 4 months of anti-TB chemotherapy, respectively, compared with patients at diagnosis. There was significant positive correlation between %NBT with plasma H2O2 and NO, but %LM was negatively correlated with plasma H2O2 in this group. CONCLUSION: The intracellular killing aspect of innate cellular immunity is deficient in patients with TB, especially 2 to 4 months after commencement of treatment. Therefore, measures (eg, arginine supplementation) to improve intracellular killing in these patients is advocated. Moreover, %LM assay with Bacillus Calmette-Guérin vaccine as an antigen may be used to differentiate those newly diagnosed patients from those on anti-TB chemotherapy.

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.

Fine characterization of glucosylated human IgG by biochemical and biophysical methods.

Nonenzymatic glycosylation of proteins finally generates advanced glycation end products (AGEs). The Schiff's base and Amadori adduct are stages of early glycation. AGE-modified IgG may undergo conformational alterations and the final entity of the process may be involved in the pathogenesis of Rheumatoid Arthritis (RA). In this study, glycation of human IgG was carried out with varying concentrations of glucose. Effect of incubation period on glycation of IgG has also been studied. Amadori adduct was detected by nitroblue tetrazolium (NBT) dye. The glucose mediated structural alterations in IgG were studied by UV, fluorescence, CD, FT-IR, DLS and DSC spectroscopy, and SDS-PAGE. Glycation-induced aggregation in AGE-IgG was reported in the form of binding of thioflavin T and congo red. Furthermore, AGE-modified IgG exhibited hyperchromicity, decrease of tryptophan fluorescence accompanied by increase in AGE specific fluorescence, loss of ß-sheet, appearance of new peak in FT-IR, increase in hydrodynamic size and melting temperature. SDS-PAGE results showed decrease in the band intensity of glycosylated-IgG compared to native IgG. Glycation-induced modifications and aggregation of IgG might be important in the pathogenesis of RA.

In vitro enzyme-mimic activity and in vivo therapeutic potential of HSJ-0017, a novel Mn porphyrin-based antioxidant enzyme mimic.

Manganese (III) 5, 10, 15, 20-tetrakis [3-(2-(2-methoxy)-ethoxy) ethoxy] phenyl porphyrin chloride, designated HSJ-0017, is a novel antioxidant enzyme mimic. The aim of the present study was to investigate the enzyme-mimic activity and the therapeutic potential of HSJ-0017 in free radical-related diseases. Superoxide dismutase (SOD) mimic activity was measured by the nitroblue tetrazolium chloride monohydrate reduction assay. Catalase (CAT) mimic activity was measured based on the decomposition of hydrogen peroxide. The antitumor, radioprotective and chemoprotective effects of HSJ-0017 were evaluated in H22 or S180 tumor-bearing Kunming mice. The anti-inflammatory and hepatoprotective effects were, respectively, evaluated in histamine-induced edema model and CCl4-induced hepatic damage model in Wistar rats. HSJ-0017 over a concentration range of 0.001-10 µmol/L significantly inhibited the generation of superoxide anion. Significant hydrogen peroxide scavenging activity was observed when the concentration of HSJ-0017 was higher than 0.01 µmol/L. HSJ-0017 at a dose of 3.0 mg/kg exhibited significant antitumor effect on S180 tumor xenografts, whereas no significant antitumor effect was observed in H22 tumor xenografts. HSJ-0017 at a dose of 3.0 mg/kg enhanced the antitumor effects of radiotherapy and chemotherapy, and reduced their toxicity. However, HSJ-0017 counteracted the antitumor effects of radiotherapy when administered simultaneously with radiotherapy. HSJ-0017 showed significant anti-inflammatory and hepatoprotective effects. Our results demonstrate that HSJ-0017 exhibits antioxidant, antitumor, anti-inflammatory, radioprotective, chemoprotective, and hepatoprotective effects. It is a potent dual SOD/CAT mimic.

Astaxanthin treatment confers protection against oxidative stress in U937 cells stimulated with lipopolysaccharide reducing O2- production.

Recently, astaxanthin (ASTA) studies have focused on several biological functions such as radical scavenging, singlet oxygen quenching, anti-carcinogenesis, anti-diabetic, anti-obesity, anti-inflammatory, anti-melanogenesis, and immune enhancement activities. In this study, we investigated the potential role protective of ASTA, an antioxidant marine carotenoid, in restoring physiological conditions in U937 cells stimulated with LPS (10 µg/ml). Our results show that pre-treatment with ASTA (10 µM) for 1 h attenuates the LPS-induced toxicity and ROS production. The beneficial effect of ASTA is associated with a reduction intracellular O2 (-) production by restoring the antioxidant network activity of superoxide dismutase (SOD) and catalase (CAT), which influence HO-1 expression and activity by inhibiting nuclear translocation of Nrf2. We accordingly hypothesize that ASTA has therapeutic properties protecting U937 cells from LPS-induced inflammatory and oxidative stress.

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.

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.