Nitroblue Tetrazolium Reduction Research Articles

Administrations of autochthonous probiotics altered juvenile rainbow trout Oncorhynchus mykiss health status, growth performance and resistance to Lactococcus garvieae, an experimental infection

The present study was tested how Oncorhynchus mykiss can respond to dietary supplementations of autochthonous probiotics, including Lactobacillus delbrukei subsp. bulgaricus and Lactobacillus acidophilus and Citrobacter farmeri by measuring different parameters. To address that, 300 fish weighing 19.08–32.9 g were fed by probiotics-enriched diets, containing 5 × 107 CFU g−1 for 60 days. Our results indicated that probiotics, especially L. acidophilus and L. bulgaricus are involved in enhancing the growth performance of this species as compared with the control group. Blood profile (Hemoglobin and Hematocrit) showed significant (P < 0.05) increases in probiotic fed groups compared with the control. Serum lysozyme and complement activities were higher in probiotic-fed fish while similar changes were not observed in the case of bactericidal activity and Nitroblue Tetrazolium (NBT) reduction. Better colonization of lactic acid bacteria in fish intestine was observed following L. acidophilus and L. bulgaricus administrations (P < 0.001). Digestive enzyme activities of intestine, including amylase, trypsin, lipase and alkaline phosphatase were elevated either significant or insignificant while protease activity did not act the same. All probiotic treatments led to mild or strong (P < 0.001) up-regulation of cytokine and growth gene expressions of intestine in comparison with the control group. Higher in vitro antagonist activities of L. acidophilus and L. bulgaricus against the Lactococcus garvieae were coincident with in vivo challenge test. The relative percentage of survival (RPS) was obtained 63.71 and 51.56 for L. bulgaricus and L. acidophilus, respectively, which were higher in those treated fish as compared to control fish. Our results may suggest that the probiotics, applied here, can promote growth performance by improving digestive enzyme activity, gut micro flora and growth gene expression. Up-regulation of immune regulatory proteins may increase the non-specific immune responses and bacterial resistance in this species as well.

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 48h, 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±3.2nM. 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 factor1 (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.

Immunomodulatory activities of Acacia catechu, a traditional thirst quencher of South India

BackgroundAcacia catechu has been widely used in Ayurveda for treating many diseases. Its heartwood extract is used in asthma, cough, bronchitis, colic, diarrhea, dysentery, boils, skin afflictions, sores and for stomatitis. The decoction of heartwood is used for drinking purpose in southern part of India especially in Kerala. ObjectiveThe current study was carried out to evaluate immunomodulatory effects of heartwood extracts of A. catechu in Swiss albino mice. Material and methodsIn vivo immunomodulatory activity was analyzed by hemagglutinating antibody (HA) titer, plaque forming cell assay and delayed type hypersensitivity (DTH). In vitro immunomodulatory potential of the extracts was studied using peritoneal macrophages and splenocytes from mice. Effect of extracts on phagocytic activity of macrophages was analyzed by nitroblue tetrazolium (NBT) reduction assay and cellular lysosomal enzyme assay. Anti-inflammatory activity was studied by nitric oxide (NO) assay and production of TNF-α and IL-10. ResultsA dose dependent increase in antibody titer was observed with extracts treatment. Treatment with extracts produced an enhancement in the number of antibody producing cells in the spleen. DTH reaction was significantly decreased with extracts treatment. An increased phagocytic response was shown by peritoneal macrophages on treatment with the extracts as evidenced by its effect on NBT reduction and cellular lysosomal enzyme activity. The extracts inhibited the release of pro-inflammatory cytokine TNF-α and the production of NO. IL-10 production was significantly increased after extract treatment. ConclusionThe results of the present study indicate the immunomodulatory effects of A. catechu extracts on humoral, cell mediated and non-specific immune functions.

Innate Immune System Status of Sulphur Mustard-Poisoned Iranian Veterans Three Decades after Exposure.

Sulphur mustard (SM) is an incapacitating chemical warfare agent which causes acute and chronic toxicities in different body organs of affected individuals. The aim of this study was to investigate the innate immune status of the Iranian veterans who were exposed to SM around 30 years earlier and had more than 25% disabilities. In this regard, most functional and non-functional parameters of innate immunity were evaluated in 35 veterans. Phagocytic activity, nitroblue tetrazolium (NBT) reduction assay and haemolytic complement activity (HCA) in addition to routine haematological parameters, serum protein electrophoresis, complements C3 and C4 levels were studied. Measures of haematological parameters, serum proteins, C3 and C4 were almost within the normal range. Functional experiments such as phagocytic activity, NBT reduction assay and HCA were normal as well. However, serum protein analysis revealed a fair decrease in percentages of α1 -globulin. Mean values of the parameters of innate immune system of the veterans three decades after SM poisoning were almost within the upper and lower normal limits. Reduced α1 -globulin - maybe subsequent to a chain of SM-induced genetic disorders - may have been the result of α1 -antitrypsin deficiency which may result in prevalent respiratory complications among these veterans. As a supplementary study, measurement of serum α1 -antitrypsin in SM-poisoned veterans could be beneficial. Further studies are required to prove this hypothesis. Further investigations on the evaluation of the acquired immunity parameters as the second line of defence may reveal a better understanding of SM veterans' immune system status.

Fe(III) and Cu(II) coordination polymers assembled from di/tris-phosphonic acid and auxiliary ligands：Structure, SOD-like activity and magnetic behavior

Hydrothermal reactions of (benzylazanediyl)bis(methylene)-diphosphonic acid (H4L1) or (benzene-1,3,5-triyltris(methylene))triphosphonic acid (H6L2) with Fe(III)/Cu(II) nitrate and 4, 4′-bipy or 2, 2′-bipy result in five new phosphonates, namely, [FeHL1(H2O)]n (1), {[Cu2L1(4, 4′-bipy)0.5(H2O)]·0.25H2O}n (2), [Cu2H2L2(4, 4′-bipy)(H2O)2]n (3), [CuH4L2(2, 2′-bipy)]n (4) and [Cu2(H4L2)2(2, 2′-bipy)2(H2O)4] (5), respectively. X-ray structural analysis indicates that 2 and 3 feature 3D framework structures with 3, 3, 3, 4-connected and 4, 5-connected topology; 1 and 4 display 1D chain structures, while 5 has a dimer structure. The di- or trisphosphonate ligands (H4L1 and H6L2) in the five complexes adopt different coordination modes and auxiliary ligands (4, 4′-bipy and 2, 2′-bipy) control the dimensionality of the final complexes. The superoxide dismutase (SOD) activities of 2–5 were assessed by modified nitrobluetetrazolium (NBT) assay. The IC50 values required to yield 50% inhibition of the reduction of NBT are 1.84, 2.02, 6.95 and 1.70 μM for 2–5, respectively, demonstrating better SOD-like activity than most of the Cu-SOD mimic enzymes reported in literatures. Among them, 2 and 3 have good stability and reusability under the simulative physiological conditions. The dismutation of 4 and 5 toward superoxide anion radicals can be attributed to the synergistic effect of the decomposition products of 4 and 5 containing Cu(II) and [H4L2]2- with or without 2, 2′-bipy or Cu(II) and 2, 2′-bipy, which were identified by liquid mass spectrometry (LC/MS). The magnetic properties of complexes 1–4 were also investigated, indicating the presence of magnetic exchange between metal ions. The results show that 1 and 3 exhibit antiferromagnetic interactions, while 2 and 4 show ferromagnetic interactions between metal centres.

Catechin rich butanol fraction extracted from Acacia catechu L. (a thirst quencher) exhibits immunostimulatory potential

Acacia catechu L., (Fabaceae) named as “catechu” is a plant, the decoction of heartwood of which is daily consumed as thirst quencher by a good percentage of the population in South India. The plant is mainly distributed in India and other Asian countries. It has been used in Indian traditional medicine for the treatment of asthma, bronchitis, colic, diarrhea, boils, skin afflictions, sores and stomatitis. The present investigation was aimed to study the immunomodulatory effects of different fractions of ethanol extract of A. catechu heartwood and HPLC analysis of the active fraction. Three fractions namely, butanol, chloroform and ethyl acetate were prepared from ethanol extract of A. catechu heartwood. Each of these fractions was assessed for its immunomodulatory activity. In vivo immunomodulatory activity was analyzed by sheep red blood cells (SRBC) specific hemagglutinating antibody titer, plaque-forming cell assay and delayed type hypersensitivity (DTH) reaction in Swiss albino mice. In vitro immunomodulating potential of the fractions was studied using murine peritoneal macrophages and splenocytes. Non-specific immune functions such as phagocytosis (nitroblue tetrazolium reduction assay and cellular lysosomal enzyme assay), nitric oxide (NO) production and cytokine release (TNF-α and IL-10) were studied in macrophages. In addition, splenocyte proliferation was also studied. In the in vivo experiments, butanol and chloroform fractions showed an increase in antibody titer dose-dependently. At higher dose (400 mg/kg b. w.) treatment the butanol fraction produced an enhancement in the number of plaque-forming cells (antibody producing cells) in the spleen. SRBC induced DTH reaction was significantly increased with butanol fraction in a dose-dependent manner. Peritoneal macrophages showed an increased phagocytic response on treatment with butanol fraction (100 μg/mL) as evidenced by its effect on nitroblue tetrazolium reduction and cellular lysosomal enzyme activity. All three fractions inhibited the production of NO and the release of TNF-α. Interleukin-10 production was significantly increased after treatment with butanol fraction. High-performance liquid chromatography analysis of the butanol fraction showed the presence of high concentration of catechin. The results suggested that butanol fraction of ethanol extract of A. catechu heartwood had immunomodulatory effects on non-specific, humoral, and cell-mediated immune functions. This study may be useful in validating the rationality of daily consumption of decoction of A. catechu and also its use in traditional medicine system. The study also suggests the possible use of A. catechu in the immunostimulatory herbal preparations.

Silica nanoparticles embedded with water insoluble phthalocyanines for the photoinactivation of microorganisms.

Silica nanoparticles (SiNPs) embedded with Zn(II) 2,9,16,23-tetrakis(methoxy)phthalocyanine (SiNPZnPcOCH3), Zn(II) 2,9,16,23-tetrakis(4-pyridyloxy) phthalocyanine (SiNPZnPcOPy) and Zn(II) 2,9,16,23-tetrakis(t-butyl) phthalocyanine (SiNPZnPctBu) were synthesized in the nonpolar core of AOT/1-butanol/water micelles using triethoxyvinylsilane and 3-aminopropyltriethoxysilane. These SiNPs-Pc presented an average diameter of about 20-25 nm. UV-vis absorption spectra presented the characteristic Soret and Q bands of phthalocyanines embedded into the nanoparticles. Moreover, red fluorescence emission of SiNPs bearing phthalocyanines was detected in water. The SiNPs-Pc produced the photodecomposition of 2,2'-(anthracene-9,10-diyl)bis(methylmalonic acid), which was used to sense the singlet molecular oxygen O2(1Δg) generation in aqueous medium. Also, the formation of superoxide anion radical was detected by nitro blue tetrazolium reduction in the presence of NADH. Photoinactivation of microorganisms was investigated in Staphylococcus aureus and Candida albicans. In vitro experiments showed that photosensitized inactivation induced by SiNPZnPcOCH3 and SiNPZnPctBu improved with an increase of irradiation times. After 30 min irradiation, over 7 log reduction was found for S. aureus. Also, these SiNPs-Pc produced a decrease of 2.5 log in C. albicans after 60 min irradiation. In both cases, a lower photoinactivation activity was found for SiNPZnPcOPy. Studies of photodynamic action mechanism showed that the photokilling of microbial cells was protected in the presence of sodium azide and diazabicyclo[2.2.2]octane. Also, a reduction on the cell photodamage was found with the addition of D-mannitol. Therefore, the photodynamic activity sensitized by SiNPZnPcOCH3 and SiNPZnPctBu in microbial cells was mediated by a contribution of both type I and type II photooxidative mechanisms. Thus, silica nanoparticles are interesting materials to vehicle ZnPcOCH3 and ZnPctBu in aqueous media to photoeradicate microorganisms.

Purification and characterization of thermostable superoxide dismutase from Anoxybacillus gonensis KA 55 MTCC 12684

A thermostable superoxide dismutase from thermophilic bacterium Anoxybacillus gonensis KA 55 MTCC 12684 which was isolated from Manikaran hotspring of Himachal Pradesh was purified to apparent homogeneity by fractional ammonium sulphate precipitation and anion exchange chromatography. A purification factor of 33.1-fold was achieved, with the purified enzyme exhibiting specific activity of 5758.4 U/mg protein. The purified superoxide dismutase was optimally active at pH 9.0 and displayed stability over a broad pH range of 7.0–10.0 and was stable up to 70 °C. SOD was localized in polyacrylamide gel by activity staining, based on the reduction of nitroblue tetrazolium (NBT) by superoxide ion. The molecular weight of superoxide dismutase was calculated as 31 kDa by SDS-PAGE. The Km and Vmax values of purified enzyme were found to be 1.002 mM and 14,285.71 U/mg of protein respectively. Tests of inhibitors indicated that the enzyme activity was inhibited by hydrogen peroxide and potassium cyanide but not by sodium azide showing that purified SOD was Cu/ZnSod.

Oxidative modifications of foetal LDL-c and HDL-c lipoproteins in preeclampsia

BackgroundOxidative modifications have been observed in lipids and proteins in lipoproteins isolated from women with preeclampsia. Thus, newborns could also be susceptible to this damage directly through their mothers. In this study, we evaluated the oxidative profile of LDL-c and HDL-c lipoproteins isolated from the umbilical cord from newborns born to women with preeclampsia.MethodsThirty newborns born to women with preeclampsia and thirty newborns born to women with healthy pregnancies were included. Lipid-damage biomarkers, including conjugated dienes, lipohydroperoxides and malondialdehyde, were measured. The reduction of nitroblue tetrazolium, formation of dityrosines, and carbonylation of proteins were assessed as indicators of protein damage. The protective activity of paraoxonase-I on HDL-c particles was evaluated. The total antioxidant capacity and lipid profiles were quantified in plasma. Data were analysed using Student’s t-tests and Pearson correlation coefficients.ResultsCompared with the control group, the preeclampsia group had an increase in the percentage of lipid damage in both lipoproteins. There was an increase of 23.3 and 19.9% for conjugated dienes, 82.4 and 21.1% for lipohydroperoxides, and 103.8 and 51.5% for malondialdehyde in LDL-c and HDL-c, respectively. However, these infants did not show evident damage in protein oxidation. The activity of the enzyme paraoxonase-I was decreased by 36.2%; by contrast, the total antioxidant capacity was increased by 40% (protein) and 28.8% (non-protein).ConclusionsThe oxidative modifications that occur in HDL-c and LDL-c isolated from newborns from women with preeclampsia are mainly caused by lipoperoxidation processes related to evident paraoxonase-I inactivation. The absence of protein damage is likely linked to an increase in total antioxidant capacity. Therefore, antioxidant support could be helpful in reducing oxidative stress in mother/newborn dyads.

The Effect of Administration of Silver Nanoparticles on the Immune Status of Chickens

Abstract The purpose of the study was to verify the hypothesis that there are doses of silver nanoparticles (Ag-NPs) that improve immune defence in chickens without compromising their health. To verify this hypothesis, an attempt was made to determine which doses of Ag-NPs (5 nm) consumed by chickens as a result of administration of hydrocolloids during varying time periods at a concentration of 5 or 10 mg Ag/l stimulate immune defence. The experiment was performed on 296 chickens assigned to 3 treatments. Chickens from the control treatment received drinking water without experimental additives. Chickens from the T-5 and T-10 treatments received a hydrocolloid of Ag-NPs at a concentration of 5 mg/l (treatment T-5) or 10 mg/l (treatment T-10) from their second week of life. Blood for analysis was collected at the age of 42 days from 8 birds per treatment. Ceruloplasmin (Cp), leukocyte count (WBC), erythrocyte sedimentation rate (ESR), interleukin IL-6, immunoglobulins IgA, IgY, phagocytic cells (% PC), phagocytic index (PI), nitroblue tetrazolium reduction (NBT), and lysozyme content in the blood was determined. Application of Ag-NPs at a concentration of 5 mg/l or 10 mg/l in the dose range of 2.87-12.25 mg/bird (administration of Ag-NPs in concentration 5 mg/l in weeks: 2; 2 and 3; 2 and 4; 2 and 5; 2 and 6 or concentration 10 mg/l in week 2) resulted in an immunostimulatory effect expressed as an increase in heterophil respiratory burst and an increased concentration of lysozyme. Higher doses of Ag-NPs exerted a pro-inflammatory effect, as indicated by elevated levels of IL-6 and ceruloplasmin, as well as a high ESR. They also stimulated B lymphocytes to produce IgA and IgA immunoglobulins.

Characteristics of Eliciting Effects of Furostanol Glycosides on Cultured Yam Cells

To unravel mechanisms of elicitor action of furostanol glycosides (FGs), the formation of superoxide anion after the addition of FGs to a suspension culture of yam (Dioscorea deltoidea Wall. ex Griseb) cells was studied. The substantial increase in superoxide level, evaluated by nitroblue tetrazolium (NBT) reduction to formazan, was found at the exponential phase of cell growth. The involvement of NADPH oxidase in the superoxide generation was revealed by means of inhibitory analysis. Diphenyliodonium chloride (DPI), the inhibitor of NADPH oxidase, compromised the action of FGs. Meanwhile, the elimination of apoplastic peroxidase did not affect the accumulation of formazan, which suggests the involvement of NADPH oxidase but not peroxidase in the superoxide generation. In addition to NBT-test, the superoxide formation was judged by changes in activity of superoxide dismutase (SOD). Exogenous FGs activated the enzyme due to the increased production of superoxide anion. In this case, DPI decreased SOD activity that conforms to the NADPH oxidase involvement in the superoxide generation. The analysis of antioxidant activity of FGs by inhibition of radicals of 2,2-diphenyl-1-picrylhydrazyl showed that FGs are weak reductants in comparison with ascorbic acid. The results of the work allow for the suggestion that, supposing a weak reducing capacity of FGs, the special feature of their exogenous action on cultured yam cells is the increase in the level of superoxide anion radical mainly produced by NADPH oxidase.

Silver nanoparticle-mediated cellular responses in isolated primary Sertoli cells in vitro

The present study explored the mechanism of cytotoxic and genotoxic effects of AgNPs on a primary culture of mouse Sertoli cells in vitro. To understand the possible molecular mechanisms of testicular lesions following exposure to AgNPs, isolated Sertoli cells were exposed to 5, 10, or 15 μg/ml. DNA damage in the Comet assay and apoptosis in the TUNEL assay were evaluated. The mRNA expression of p53 and bcl-2 genes and their proteins involved in apoptosis was also investigated. The antioxidant status of treated Sertoli cells was determined by measuring superoxide dismutase (SOD-1), catalase (CAT) and glutathione peroxidase (GPX-1) using quantitative polymerase chain reaction (qPCR). The superoxide anions were detected using the nitroblue tetrazolium (NBT) reduction assay. Results indicated that AgNP exposure causes increased oxidative stress levels. The activation of p53, repression of bcl-2 and reduction of endogenous antioxidant enzymes were also involved in these mechanistic pathways, leading to reduced cell numbers and cell detachment.

Evaluation of immunotropic activity of gold nanocolloid in chickens

Gold nanoparticles (AuNPs) are one of the most examined nanomaterials, but information about their immunogenic potential is still insufficient. Understanding interaction of AuNPs with immune system is essential in designing their safety and possibilities of biomedical applications. An experiment was conducted to determine immunotropic activity of gold nanocolloid (AuNPs) administered orally to chickens depending on dose and duration time. 162 birds were assigned to 9 experimental groups of 18 birds each. The control group (C) did not receive AuNPs. Groups: T10.5, T11.0, T11.5, T12.0, received nano-gold in a rate of 0.5 mg/kg body weight/d, 1.0 mg/kg body weight/d, 1.5 mg/kg body weight/d and 2.0 mg/kg body weight/d in 8–14, 22–28 and 36–42 days of the life. The birds in groups T20.5, T21.0, T21.5, T22.0, received nano-gold in the same doses, but only in 8–10, 22–24 and 36–38 days of life. Phagocytic activity of leukocytes was determined in vitro using Staphylococcus aureus 209P strain, their respiratory burst activity was quantified by nitroblue tetrazolium reduction test. Serum lysozyme content was determined by the turbidimetric method. The Wintrobe method was used to determine the erythrocyte sedimentation rate. Ceruloplasmin in the blood plasma was estimated by the p-phenylenediamine colorimetric method. The level of chicken immunoglobulins: IgA, IgM and IgY and interleukin IL-6 in the blood were determined using ELISA tests. The lowest dose of AuNPs, independently on duration time had no effect on immune parameters of chickens. In all other groups receiving nano-gold for a shorter period (T2), there was an increase in the respiratory burst activity of leukocytes and a drop in lysozyme activity in blood. The higher doses (1.5 and 2.0 mg/kg body weight/d) of the nano-gold administered for the longer time period had a pro-inflammatory effect, as indicated by an increase in the level of interleukin 6 and ceruloplasmin activity as well as the erythrocyte sedimentation rate. They also contributed to an elevation of class IgA and IgY contents in blood. The results of the study revealed that the influence of nano-gold on immune response of chickens were dependent both on dose and duration time. Long lasting administration of higher doses of AuNPs contributed to adverse effect in form of inflammation response. To avoid the development of inflammatory reaction, administered dose of nano-gold should not exceed 1.0 mg/kg body weight/d.

Cuscuta campestris induces apoptosis by increasing reactive oxygen species generation in human leukemic cells.

Cuscuta campestris or common dodder is a holoparasitic plant that has been valorized for treatment of liver injury and cancer prevention in traditional medicine. Recently, extract of C. campestris had shown moderate antimicrobial properties and cytotoxic effects. In this study, we examined the level of cellular oxidants, cytotoxicity, apoptosis and differentiation induced by hydroalcoholic extract of C. campestris (CCE) (12.5-200 µg/ml), as well as arsenic trioxide (As2O3, 50 µM), in human leukemic (HL60 and NB4) and normal polymorph nuclear cells after 72 hr treatment. Resazurin assay was used to determine cell viability following treatment with C. campestris. Intracellular reactive oxygen species (ROS) and apoptotic cells were measured by fluorimetry using carboxy 2', 7'-dichlorofluorescein diacetate and propidium iodide (PI), as staining reagents, respectively. The differentiation of leukemic cells was evaluated by Giemsa staining and nitro blue tetrazolium (NBT) reduction. C. campestris inhibited cell viability with IC50 values of 23.9 µg/ml for HL60 and 60.3 µg/ml for NB4 cells after 72 hr treatment. ROS formation was also concentration-dependently increased following treatment with C. campestris. In addition, the number of apoptotic cells significantly increased to 88.4% and 62.3% in CCE (200 µg/ml)-treated HL60 and NB4 cells, respectively, which was higher than that of As2O3 (50 µM)-treated leukemic cells (p<0.001). Nonetheless, C. campestris did not induce differentiation of leukemic cells towards granulocytic pattern. The present study demonstrated that C. campestris induced apoptosis through ROS production without having differential effect on leukemic cells, in concentration- and time-dependent manners. Understanding of precise signaling pathway by which C. campestris induce apoptosis, needs further research.

Effects of Fifty-Hertz Electromagnetic Fields on Granulocytic Differentiation of ATRA-Treated Acute Promyelocytic Leukemia NB4 Cells

Background/Aims: Life on Earth is constantly exposed to electromagnetic fields (EMFs) and the effects induced by EMFs on biological systems have been extensively studied producing different and sometimes contradictory results. Extremely low-frequency electromagnetic fields (ELF-EMFs) have shown to play a role in regulating cell proliferation and differentiation, although how EMFs influence these processes remains unclear. Human acute promyelocytic leukemia (APL) cells are characterized by the arrest of differentiation at the promyelocytic stage due to epigenetic perturbations induced by PML/RARα fusion protein (Promyelocytic Leukemia protein - PML/Retinoic Acid Receptor alpha - RARα). Therapeutic administration of all-trans retinoic acid (ATRA) re-establishes the leukemogenic mechanism re-inducing the normal differentiation processes. Methods: We studied the effects of ELF-EMFs (50 Hz, 2 mT) on the ATRA-mediated granulocytic differentiation process of APL NB4 cells (a cell line established from the bone marrow of a patient affected by the acute promyelocytic leukemia) by monitoring cellular proliferation and morphology, nitrob lue tetrazolium (NBT) reduction and the expression of differentiation surface markers. Finally, we investigated mechanisms focusing on reactive oxygen species (ROS) generation and related molecular pathways. Results: ELF-EMF exposure decreases cellular proliferation potential and helps ATRA-treated NB4 cells to mature. Furthermore, the analysis of ROS production and the consequent extracellular signal regulated kinases (ERK1/2) phosphorylation suggest that a changed intracellular oxidative balance may influence the biological effects of ELF-EMFs. Conclusions: These results indicate that the exposure to ELF-EMF promotes ATRA-induced granulocytic differentiation of APL cells.

Autophagy and Ubiquitin-Mediated Proteolytic Degradation of PML/Rarα Fusion Protein in Matrine-Induced Differentiation Sensitivity Recovery of ATRA-Resistant APL (NB4-LR1) Cells: in Vitro and in Vivo Studies

Background/Aims: Although the cure rate of acute promyelocytic leukemia (APL) has exceeded 90%, the relapse/refractory APL that resistant to all-trans retinoic acid (ATRA) or ATO was still serious concern. Matrine (MAT) could improve the differentiation ability of ATRA-resistant APL cells. This study aimed to explore how the APL-specific fusion protein was degraded in ATRA-resistant APL with the application of MAT and ATRA. Methods: ATRA-sensitive (NB4) and ATRA-resistant (NB4-LR1) cell lines were used. Nitroblue tetrazolium reduction assay and flow cytometry were used to detect the differentiation ability. The activity of ubiquitin-proteasome and autophagy-mediated pathways in both cells treated with ATRA with or without MAT were compared in protein and mRNA level (Western blot analysis, qRT-PCR), the Fluorescent substrate Suc-LLVY-AMC detection was used to detect the activity of proteasome, and electron microscope for observing autophagosome. MG 132(proteasome inhibitor), rapamycin (autophagy activator), hydroxychloroquine (lysosomal inhibitor) and STI571 [retinoic acid receptor alpha (RARα) ubiquitin stabilizer] were used as positive controls. The effect of MAT was observed in vivo using xenografts. Results: MAT improved the sensitivity of NB4-LR1cells to ATRA treatment, which was consistent with the expression of PML-RARα fusion protein. MAT promoted the ubiquitylation level in NB4-LR1. MG 132 induced the decrease in RARα in both cell lines, and hampered the differentiation of NB4 cells. MAT also promoted the autophagy in NB4-LR1 cells, with an increase in microtubule-associated protein 1 light chain3 (LC3)-II and LC3-II/LC3-I ratio and exhaustion of P62. The expression of LC3II increased significantly in the MAT and ATRA + MAT groups in combination with lysosomal inhibitors. A similar phenomenon was observed in mouse xenografts. MAT induced apoptosis and differentiation. Conclusions: Autophagy and ubiquitin-mediated proteolytic degradation of PML/RARα fusion protein are crucial in MAT-induced differentiation sensitivity recovery of NB4-LR1 cells.

Ferula gummosa gum induces apoptosis via ROS mechanism in human leukemic cells.

Ferula species known for its oleo-resins that are recognized valuable industrial crops and food products. In this study, we examined the level of cellular oxidants, cytotoxicity, apoptosis and differentiation induced by oleo resin gum from Ferula gummosa (30-250 µg/mL), as well as Arsenic trioxide (50 µM, as positive control), in leukemic (NB4 and HL-60 cells) and normal polymorph nuclear cells during 72 h. Resazurin assay was used to determine cell viability following treatment with F. gummosa (30-250 µg/mL). Intracellular reactive oxygen species was measured by fluorimetry using carboxy 2', 7'-dichlorofluorescein diacetate. Apoptotic cells were evaluated using PI staining of DNA fragmentation by flow cytometry (sub-G1 peak). Differentiation of cells evaluated by Giemsa staining and Nitro Blue Tetrazolium reduction. F. gummosa showed a concentration-dependent suppression in cell survival with IC50 values of 41.8 µg/mL for HL60 and 59.2 µg/mL for NB4 cells after 72 h treatment. ROS formation and apoptotic cells were concentration-dependently increased following treatment with F. gummosa, similar to As2O3. F. gummosa did not induce differentiation of leukemic cells towards granulocytic pattern. The resin did not have toxic effect on PMN cells (&lt;800 µg/mL). In conclusion, the present study demonstrated that F. gummosa induced apoptosis through ROS mechanism on leukemic cells as a concentration and time dependent manner. The precise signaling pathway by which F. gummosa induce apoptosis needs further research.

Antioxidant Enzyme Activities of some Brassica Species

This paper set out to comparatively study five species: white cabbage (Brassica oleracea L. var. capitata alba Alef.), red cabbage (Brassica oleracea L. var. capitata f. rubra Alef.), Kale (Brassica oleracea L. var. Acephala), cauliflower (Brassica oleracea var. botrytis) and broccoli (Brassica oleracea var. cymosa) in order to identify those with high enzymatic and antioxidant activities. The enzymatic activity of superoxide dismutase (SOD), catalase (CAT) and soluble peroxidase (POX) as well as the antioxidant activity against 2.2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical cation were determined. Total superoxide dismutase activity was measured spectrophotometrically based on inhibition in the photochemical reduction of nitroblue tetrazolium. Total soluble peroxidase was assayed by measuring the increase in A436 due to the guaiacol oxidation and the catalase activity was assayed through the colorimetric method. The capacity of extracts to scavenge the ABTS radical cation was assessed colorimetric using Trolox as a standard. The obtained results show that studied enzymatic activities and the antioxidant activity against ABTS vary depending on the analyzed species. So, among the studied Brassicaceae species, it emphasize red cabbage with the highest enzymatic activity (CAT 22.54 mM H2O2/min/g and POX 187.2 mM ΔA/1min/1g f.w.) and kale with highest antioxidant activity, of 767 μmol TE/100g f.w. The results of this study recommendintroducing the studied varieties in diet due to the rich antioxidant properties.

The efficacy of Poly-β-Hydroxy Butyrate (PHB)/biosurfactant derived from Staphylococcus hominis against White Spot Syndrome Virus (WSSV) in Penaeus monodon

White Spot Syndrome Virus (WSSV) is one of the most important causative agents of Penaeid shrimps diseases that incur heavy losses to the shrimp aquaculture. It has severe impact on the sustainability and the production of Penaeus monodon. Hence, the present study focussed on the investigation of Poly-β-hydroxybutyrate/biosurfactant as immunostimulants against WSSV infected shrimps. Infection of WSSV was periodically checked in all the experimental shrimps using PCR diagnostic kit. After ensuring all shrimps were free of viral infection, experiments were carried out to analyze the nonspecific immune responses (prophenol oxidase, nitro blue tetrazolium reduction assay and total haemocyte count) both in control and experimental group. Further, gills and muscles of Penaeus monodon were subjected to proteome analysis after treated it with PHB/biosurfactant independently in the concentration of 2% and 5% each. Increase in the level of haemocytes was observed in both PHB (26 ± 2 × 10⁴ cells)/biosurfactant (28 ± 2 × 104 cells) treated shrimps, when compared with control (17 ± 2 × 10⁴ cells). proPhenolOxidase (proPO) activity was also enhanced in treated groups compared to WSSV infected shrimps. Less production of superoxide anion was observed in control and treated groups. Differences in the protein expression was analyzed in muscle tissue of control, WSSV infected and PHB/biosurfactant treated shrimps. Our finding suggested that partial substitution of feed with 2% PHB and biosurfactant showed increased rate on the survival of WSSV infected P. monodon which might be due to either the over expression/down regulation of proteins that play a vital role in enhancing the immune system/the progression of the disease respectively.

CD4+ count and Nitro-Blue Tetrazolium reduction rate of neutrophil in newly diagnosed HIV-infected adults in Sokoto Metropolis

Human immunodeficiency Virus (HIV) is a major public health problem in sub-Saharan Africa. It is known to cause malfunction of the immune cells thereby predisposing to profiles of morbidity and fatality. The current study evaluated the functional activity of neutrophils and lymphocyte sub set CD 4 + count) in newly diagnosed HIV – infected adults in Sokoto, Nigeria. A total of 126 male and female adults were recruited for the study, comprising of 64 newly diagnosed HIV– infected subjects and 62 age-and sex-matched apparently healthy individuals as controls. CD 4 + cells were enumerated using flow Cytometric method and neutrophil phagocytic activity was determined using Nitro- Blue Tetrazolium (NBT) Reduction test. Data was analyzed using Microsoft Excel and independent sample t-test for comparison of means. P-value less than or equal to 0.05 (P ≤ 0.05) was regarded as statistically significant. The CD 4 + count and neutrophil ingestion rate of NBT were significantly (P = 0.000) lower in newly diagnosed HIV- infected subjects compared with the values in control. The CD 4 + count and formazan generated by neutrophil were significantly (P = 0.024 and 0.012 respectively) higher in newly diagnosed HIV– infected female compared with male subjects. The decreased CD 4 + count and neutrophil phagocytic activity in ART-naive HIV- infected subjects is an indication that HIV suppresses both innate and adaptive arms of immune response. However, this effect was more pronounced in male subjects. The possible mechanism for this gender differences are discussed. Keywords : Neutrophil phagocytosis, CD 4 + , NBT, HIV, Sokoto.